Multi-analyte diagnostic system and computer implemented process for same

ABSTRACT

A multi-analyte diagnostic system for use with a computer. The diagnostic system includes a flow analyzer including a co-planar light source-optical detector array, the flow analyzer being communicatable with the computer. The diagnostic system also includes a memory medium readable by the computer and storing computer instructions. The instructions include the following steps. A biological sample is run through the flow analyzer. The identity and quantity of at least one analyte of interest in the biological fluid is determined substantially simultaneously to the sample-running step.

This application is a continuation of, and claims benefit under 37U.S.C. § 120 of U.S. patent application Ser. No. 09/311,115 filed May13, 1999 now U.S. Pat. No. 6,592,822, which is incorporated by referencein its entirety, which claims benefit under 37 U.S.C. § 119(e) based onU.S. Provisional Application No. 60/085,981, filed May 14, 1998, hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention pertains generally to a diagnostic system and/ormethod, and more particularly to a substantially simultaneous andmultiplexed, multi-analyte diagnostic system and/or method forperforming assays using a flow analyzer.

BACKGROUND OF THE INVENTION

Flow cytometry utilizes an optical technique that analyzes particles ina fluid mixture based on the particles' optical characteristics using aflow cytometer. Background information on flow cytometry is, forexample, found in Shapiro, Practical Flow Cytometry, Third Ed. (Alan R.Liss, Inc. 1995), incorporated herein by reference. Conventional flowcytometers have been commercially available since the early 1970s andpresently cost, for example, more than $120,000. They can be behemothsin size, occupying upwards of 13 cubic feet and weighing well over 200pounds.

In conventional flow cytometers, as shown in FIGS. 1 and 2, sample fluidcontaining sample cells or microspheres having reactants on theirsurfaces is introduced from a sample tube into the center of a stream ofsheath fluid. The sample fluid stream is injected into, at, or near, thecenter of the flow cell or cuvette. This process, known as hydrodynamicfocusing, allows the cells to be delivered reproducibly to the center ofthe measuring point. Typically, the cells or microspheres are insuspension in the flow cell.

A continuous wave laser 1900 focuses a laser beam on them as they passthrough the laser beam by a flow of a stream of the suspension. Lasersin conventional flow cytometers often require shaping a round beam intoan elliptical beam to be focused on the flow cell. As shown in FIG. 2,this elliptical beam is often formed from the round beam using a beamshaping prismatic expander 1960 located between the laser and the flowcell. When an object of interest 1905 in the flow stream is struck bythe laser beam, certain signals are picked up by detectors. Thesesignals include forward light scatter intensity and side light scatterintensity. In the flow cytometers, as shown in FIGS. 1 and 2, lightscatter detectors 1930, 1932 are located opposite the laser (relative tothe cell) to measure forward light scatter intensity, and to one side ofthe laser, aligned with the fluid-flow/laser beam intersection tomeasure side scatter light intensity.

In front of the forward light scatter detector 1930 can be an opaque bar1920, called a beam stop, that blocks incident light from the laser.Thus, the beam stop ensures that as little of the beam as possible willinterfere with the measurement by the forward light scatter detector ofthe relatively small amount of light which has been scattered, by theflow cell, at small angles to the beam. Forward light scatter intensityprovides information concerning the size of individual cells, whereasside light scatter intensity provides information regarding the relativesize and refractive property of individual cells.

Known flow cytometers, such as disclosed in U.S. Pat. No. 4,284,412 toHANSEN et al., incorporated herein by reference, have been used, forexample, to automatically identify subclasses of blood cells. Theidentification was based on antigenic determinants on the cell surfacewhich react to antibodies which fluoresce. The sample is illuminated bya focused coherent light and forward light scatter, right angle lightscatter, and fluorescence are detected and used to identify the cells.

As described in U.S. Pat. No. 5,747,349 to VAN DEN ENGH et al.,incorporated herein by reference, some flow cytometers use fluorescentmicrospheres, which are beads impregnated with a fluorescent dye.Surfaces of the microspheres are coated with a tag that is attracted toa receptor on a cell, an antigen, an antibody, or the like in the samplefluid. So, the microspheres, having fluorescent dyes, bind specificallyto cellular constituents. Often two or more dyes are usedsimultaneously, each dye being responsible for detecting a specificcondition.

Typically, the dye is excited by the laser beam from a continuous wavelaser 1900, and then emits light at a longer wavelength. As shown inFIG. 1, dichroic filters 1940 split this emitted light and direct itthrough optical detectors 1950, 1952, 1954 that can be arranged relativeto the laser. The optical detectors 1950, 1952, 1954 measure theintensity of the wavelength passed through a respective filter. Thefluorescence intensity is a function of the cells' absorption offluorescent dye.

FIG. 2 depicts a prior art flow cytometer which uses beam splitters1942, 1944, 1946 to direct light from the flow cell 1910 tophoto-multiplier and filter sets 1956, 1958, 1959 and to side lightscatter detector 1932. This flow cytometer employs a mirror 1970 toreflect forward light scatter to forward light scatter detector 1930.

However, I have determined that the properties of the fluorescent dyesthemselves limit this flow cytometric technique to about three differentwavelengths. The difference in energy, and hence wavelength, between anexcitation photon and emission photon is known as Stokes shift.Generally, the larger the Stokes shift from the excitation wavelength,the broader and weaker the emission spectra.

At any given excitation wavelength, I have determined that there areoften only a limited number of dyes that emit a spectrum of wavelengthsnarrow enough and sufficiently separated enough that they areindividually measurable simultaneously. Of these, there are fewer dyesstill that exhibit good quantum efficiency, for example, between 5 and40%. Other values for quantum efficiency are also acceptable. Forexample, values of 75 to 80% are acceptable. Consequently, researchersin flow cytometry and other fields have been limited to roughly threefluorescent labels, namely, for green, yellow-orange, and red light.

The limitation on the number of fluorescent labels necessarily crimpsthe amount of analysis that can be done on any one sample. Therefore,for meaningful analysis, a larger quantity of sample is required andmore runs of the sample through the flow cytometer must be performed.This necessarily increases the time needed to analyze the sample.However, time is often not available in an emergency room environment,for example, where a small blood sample must be screened simultaneouslyfor many diagnostic indicators, including therapeutic and abused drugs,hormones, markers of heart attack and inflammation, and markers ofhepatic and renal function. In addition, for efficiency reasons, it isdesirable to minimize the testing time to increase the number of teststhat can be performed over a predetermined time interval.

One way to overcome the limitation on the number of fluorescent labels,I have determined, is to use two lasers of different frequencies, eachfocused on a different spot along the flow stream. Such a configurationis called a multi-station flow cytometer. As a particle passes a firstlaser, up to three fluorescence measurements are taken. Then, as theparticle passes the second laser, up to three more measurements aretaken using a time-gated amplifier at a predetermined time intervalafter signals have been detected at the upstream observation point. FIG.3 illustrates this method.

It should be noted that the upper pair of particles A, B show the lowerpair of particles A, B at a later time as the particles progress upwardthrough the flow cell; the particles themselves are the same. In thiscase, laser #1 strikes particle A. A detector for Laser #2 must wait fora particle to pass through the beam of Laser #2.

Despite this dual laser approach, I have determined that it is oftenimpossible to know for certain whether the measurements are made on thesame particle. Because the measurement events at the sets of detectorsare separated temporally and spatially, I have discovered that, besideslaser emission timing problems, even the slightest flow turbulence canmix particles in suspension, thereby increasing the likelihood thatsubsequent measurements are not made on the same particle as theprevious measurements.

Further, particles in the sample fluid exhibit different velocities asthey pass through the flow cell depending on their respective distancesfrom the center of the sample fluid flow stream. Plainly, a an particlecloser to the center would travel faster than a particle further awayfrom the center. As such, it is difficult or impossible to be sureexactly when a particle detected by a detector for Laser #1 will passthrough a beam of Laser #2.

Referring to FIG. 3, flow turbulence, for example, causes particle B tochange places with particle A such that laser #2 strikes particle B,instead of particle A. By extension, this unacceptable problem compoundsas lasers and detectors are added to the device.

Despite this flaw, such multiple illumination beam capabilities havebeen limited to expensive, complex sorters and are not typically foundin smaller, less expensive instruments. Besides being large andexpensive, such machines are often fully burdened in the clinicalsetting with CD4-CD8 lymphocyte analysis.

Compounding the above-mentioned shortcomings of existing devices andmethods, I have discovered that existing methods of data collection andanalysis thereof is tedious, slow, and non-real-time. That is,substantially simultaneous detection of multiple analytes, or ofseparately identifiable characteristics of one or more analytes, throughsingle-step assay processes is presently not possible, or to the extentpossible, has provided limited capability and thus has yieldedunsatisfactory results. Reasons for these disappointing results includethe following. First, the length of time typically required to enabledetection and classification of multiple analytes is unacceptably long.Second, the prior art assays exhibited low analyte sensitivities, whichoften lead to significant analytical errors and unwieldy collection,classification, and analysis of prior art algorithms relative to largeamounts of collected data.

An existing bead set separation method involves the following steps.First, a test tube having sample fluid and sets of reporter beads mustbe loaded into the flow cytometer and depress the “Acquire” button.Second, when the desired number of data events have been collected, the“Stop” button must be pressed. Third, a file containing the collecteddata must be saved to a hard drive of a computer. Fourth, a control andanalysis software package must be opened. Fifth, the file must be loadedinto the control and analysis software package. Sixth, an x-y plot ofFL2 v. FL3 must be charted, where FL2 and FL3 are orange and redfluorescence classification parameters for the sets of beads. Seventh,the sets of beads in the plot, represented by clouds of dots, must bevisually located and a polygon gate must be drawn around the first setof interest to eliminate stray data points. Eighth, the file must befiltered for events that fall within the polygon gate. Ninth, thestatistics must be displayed and the mean value of FL1 must be noted,wherein FL1 is the green fluorescence measurement for the analyte ofinterest.

Tenth, FL1 to FL2 percent spill-over must be calculated and subtractedfrom the mean value of FL2 to correct the value of FL2. Eleventh, thecorrected value of FL2 is used to look up manually which bead set waslocated in the polygon gate. Twelfth, the FL2 to FL1 percent spill-overis calculated and subtracted from the mean value of FL1. Thirteenth, theassay result is determined from the adjusted value of FL1. The previousthirteen steps are manually repeated for each remaining set of beads.

In addition to the tedium associated with the above-described bead setseparation method, I have discovered that the subjectivity associatedwith estimating the boundaries of the polygon gates is unacceptable. Thevalue of any assay using this method depends largely on the variablejudgment of a lab technician. It is often impossible to separate somesets of beads because of overlap of bead regions on the FL2-FL3 plot.Moreover, because of FL1 to FL2 spill-over, the FL2 value of a subsetincreases sufficiently to overlap with other fluorescence values ofother bead sets. Consequently, because of the spill-over, two subsetsoccupy substantially the same region, making them impossible todistinguish visually there between. The net result of these difficultiesis the inability to determine during a sample run, the existence andquantity of an analyte of interest.

In view of the above, I have determined that it would be desirable tohave a system and/or method for detecting multiple analytes in a fluidsample by flow cytometric analysis and for analyzing and presenting thedata in real-time.

I have determined that it would be desirable to have such a systemand/or method, which eliminates the variability of human judgment andsubjectivity from the data collection and analysis by performing datacollection, bead set classification, and analysis techniques all carriedout substantially simultaneously or contemporaneously.

I have also determined that it would be desirable to have such a systemand/or method using a flow analyzer that is a fraction of the size,weight, and cost of conventional flow cytometers. That is, I havedetermined that the current “mainframe-style” flow cytometer must bereplaced by a “desktop-style” personal cytometer.

I have further determined that it would be desirable to have such asystem that is many times as fast as conventional flow cytometers andyet requires a fraction of the sample volume demanded by theconventional flow cytometers.

I have also recognized a deficiency in the current approach to signalprocessing in flow cytometry, which uses peak detectors to measure anevent. When a peak is found, the peak detectors are disabled while thepeaks are measured and processed. “Dead time,” the time period duringwhich events can pass through the laser focal point undetected, ishighly problematic when the flow cytometer is being used to search forrare events.

Prior art methods, such as U.S. Pat. No. 5,550,058 to Corio et al.,incorporated herein by reference, are largely unsuccessful. However, noknown prior art method and/or system, including that of Corio et al.,has reduced dead time to zero. For example, Corio et al. pre-qualifiesan event electronically to reduce the chance that a rare event slips byduring dead time. The Corio et al. system sorts particles at a selectedyield/purity ratio which ratio can include an intermediate value of themaximum yield and the maximum purity.

Prior art systems and/or methods, which do not use peak detection, usean integrator to measure the area under the pulse. Again, events passthrough the laser beam undetected while the measurement is made. Thus,use of an integrator also fails to reduce dead time to zero.

In view of the above-described dead time problem, I have determined thatit would be desirable to have a system and/or method for detectingmultiple analytes in a fluid sample that reduces dead time in flowanalysis to zero.

SUMMARY OF THE INVENTION

It is, therefore, a feature and advantage of the instant invention toprovide, a system and/or method for detecting multiple analytes in afluid sample by flow cytometric analysis and for analyzing andpresenting the data in real-time.

It is also a feature and advantage of the present invention to providesuch a system and/or method, which eliminates the variability of humanjudgment and subjectivity from the data collection and analysis byperforming data collection, bead set classification, and analysistechniques substantially simultaneously or contemporaneously.

It is another feature and advantage of the instant invention to providesuch a system and/or method using a flow analyzer that is a fraction ofthe size, weight, and cost of conventional flow cytometers. That is, Ihave determined that to deliver the maximum benefit of the instantdiagnostic system to the greatest number of users, the current“mainframe-style” flow cytometer must be replaced by a “desktop-style”personal cytometer.

It is also a feature and advantage of the present invention to providesuch a system that is many times as fast as conventional flow cytometersand yet requires a fraction of the sample volume demanded by theconventional flow cytometers.

It is also a feature and advantage of the present invention to providesuch a system and/or method that reduces dead time to zero. For example,the system and/or method include constant fixed rate over sampling wheresignal samples are continuously stored at a predefined interval. Byusing a second thread to analyze the contents of the circular buffer andprocess the events, events are never missed, and, hence, there is nodead time.

To this end, it is a feature and advantage of the instant invention toprovide a system including a flow analyzer that is approximatelyone-eighth the size, weight, and cost of most conventional flowcytometers. The system, optionally, is approximately eight times asfast, and, for example, requires one-eighth the sample volume. Thesystem, optionally, is modular to facilitate easy on-site repair andcomponent upgrade. Optionally, it is also controlled by anindustry-standard serial or parallel interface, allowing the system torun on a variety of, for example, personal computer environments and toform laptop or desktop factors, under the direction of, for example, auser-friendly graphical user interface.

By achieving the specifications described above, the instant inventionprovides heretofore uncommon applications for multi-analyte diagnosticsystems, ranging from a large clinical laboratory to small point-of-carefacility. That is, the speed and technical elegance of the system makeit well-suited to, for example, an emergency room environment, where asmall blood sample, for example, is screened simultaneously for manydiagnostic indicators. Such indicators, for example, include therapeuticand abused drugs, hormones, markers of heart attack and inflammation,and/or those of hepatic and renal function. The small size, low cost,and quiet operation of the system allows placement thereof in, forexample, virtually every blood bank. Donors at such an equipped bloodbank can be tested instantly for blood type and transmissible infectiousdiseases, thereby advantageously avoiding the collection of blood unitsdestined for rejection. Additionally, the small sample volumes processedby the instant system bring the power of multi-analyte testing to, forexample, neonatal and pediatric clinics, often advantageously performingcomplex analyses for less than the cost of a single analyte conventionaltest.

In an exemplary embodiment, the instant multi-analyte diagnostic systemperforms real-time bioassays using, for example, multiple classes ofmicrospheres. Each microsphere in a class is coated with a reactantunique to that class. Each class, for example, serves to assay for arespective analyte of interest. Alternatively, more than one class ofmicrospheres, for example, serves to assay for the same analyte ofinterest. The classes, optionally, are distinguishable by fluorescentlabels and/or size so that each class has a respective color and/or sizesignature. Thus, using the multiple classes of microspheres, multipleanalytes, for example, are assayed simultaneously.

The reactants of these assays, for example, are anchored or secured tothe surface of the above-mentioned uniquely fluorescent microspheres.Each assay includes at least one microsphere, and preferably up to athousand or more microspheres. Thus, for example, to conduct one hundredassays, the instant invention includes, for example, one hundreddistinguishable classes of microspheres, totaling, for example, 100,000microspheres. The instant invention, for example, individually analyzeseach microsphere in a flow stream at a rate of up to 20,000 or morebeads per second, accurately classifying each to its own unique class orsubset based on its fluorescent color and/or size signature.Additionally, the instant invention scans each microsphere for thepresence of a color, different from those used to provide classsignatures, that quantifies the assay occurring at the surface of eachmicrosphere.

By way of illustration, application of the instant invention is, forexample, found in an allergist's office. An allergist, for example,screens a patient for various allergic sensitivities. Current methodsrequire that a patient's blood sample be sent from the office to a largeclinical laboratory, or that a standard “scratch” test be performed on apatient's skin. Plainly, waiting for blood test results from a largeclinical laboratory necessarily limits immediate patient care.

Skin testing patients, using the “scratch” test, is used for suspectedimmediate-type hypersensitivity to one or more environmental substances.The test is performed by placing a drop of allergen(s) on the skin andmaking a needle prick through the drop(s) and into the underlyingepidermis. Puncture sites are examined over the next 20 minutes for awheal and flare skin response which, if present, indicatesantibody-mediated (IgE) hypersensitivity to the test allergen. Thescratch test is subject to an unacceptable rate of false-positives,false-negatives, and limited sensitivity.

In contrast, the instant invention, optionally, incubates, for example,a single drop, or more than a drop, of patient blood for less thanfifteen minutes, between fifteen to thirty minutes, or greater thanthirty minutes. Then, running the incubated sample through the instantinvention in a matter of seconds, the diagnostic system provides ahighly accurate, quantitative analysis, and if desired, a qualitativeanalysis, of hypersensitivity to, for example, the sixty-four allergenssimultaneously or substantially simultaneously. In these assays, thereagent or reactant used is, for example, 0.1% or less than thatrequired for a conventional enzyme linked immunosorbent assay (ELISA)format.

More specifically, the instant invention provides a multi-analytediagnostic system for use with a computer. The diagnostic system, forexample, includes a flow analyzer including a substantially co-planaroptical assembly having at least one light source and at least oneoptical detector. The flow analyzer is, optionally, communicatable withthe computer. The diagnostic system, optionally, also includes a memorymedium readable by the computer and storing computer instructions. Theinstructions, for example, include the following sequential,non-sequential, or independent steps. A biological sample, for example,is run through, or processed using, the flow analyzer. An identity andquantity of one or more analytes of interest in the biological sample,for example, is determined substantially simultaneously to the runningor processing step. The one or more light sources optionally include aplurality of light sources and the one or more optical detectorsoptionally include a plurality of optical detectors. The plurality oflight sources includes identical, similar, or overlapping focal regions.The plurality of light sources, for example, includes a plurality oflaser diodes emitting continuous wave light. The plurality of laserdiodes optionally includes laser diodes emitting a plurality ofwavelengths of continuous wave light. Optionally, the laser diodesinclude one or more diode pumped lasers, such as YAG lasers. The flowanalyzer, optionally, includes a cuvette having a flat air-to-glassinterface relative to each light source and relative to each opticaldetector. The cuvette, optionally, includes a cuvette having a hexagonalcross-section. Optionally, the cuvette includes a substantially flatglass-to-fluid interface. The cuvette optionally includes a neck regionhaving one of an internal rectangular cross-section and an internalsquare cross-section.

The one or more light sources, optionally, include two light sources.Each light source, optionally, emits respective two distinct wavelengthsof light. The one or more optical detectors, optionally, includes fouroptical detectors.

The flow analyzer, optionally, includes a multi-pass filter or aplurality of bandpass filters optically coupled in parallel to one ormore optical detectors via a respective multi-mode cable. The flowanalyzer, optionally, includes, for each band-pass filter, a standardamplifying photo-detector and a standard analog-to-digital converterconnected in series thereto. The amplifying photo-detector includes astandard photomultiplier tube, a standard avalanche photo-diode, or astandard p-i-n photo-diode. The flow analyzer, for example, includes foreach band-pass filter, an optional standard inverting amplifier inseries with a standard low pass Nyquist filter, connected between theamplifying photo-detector and the analog-to-digital converter.

The flow analyzer, optionally, includes one or more magnification lensfor magnifying light emission or reflection from the cuvette. Forexample, the magnification lens may include a lens having amagnification of up to or more than 15×. Advantageously, themagnification lens obviates use of the above-mentioned multi-mode cableor optical fiber. For example, the flow analyzer optionally includes amirror reflecting the light from the cuvette to appropriate detectors.

The diagnostic system optionally includes a digital interface board inthe flow analyzer and connectable to the computer via a serial orparallel interface. The digital interface board, optionally, includes astandard microcontroller in communication with the flow analyzer, and astandard digital signal processor in communication with themicrocontroller and each analog-to-digital converter. The digital signalprocessor, optionally, includes a standard circular memory buffer havinga first movable pointer, a second movable pointer, and a plurality ofstorage positions. The first pointer, optionally, points to an oldeststorage position into which new sample data can be stored. The secondpointer, optionally, points to a storage position from which the digitalsignal processor is to read the next sample data to be analyzed. Theflow analyzer includes a cuvette, a sample pump communicating with themicrocontroller and connected to the cuvette, and a sheath fluidreservoir communicating with the microcontroller and connected to thecuvette. The flow analyzer, optionally, includes a waste receptacle. Themicrocontroller, upon assay completion, optionally, communicates withthe sample pump to halt sample fluid flow or divert any remaining sampleto the waste receptacle, and, optionally, communicates with the sheathfluid reservoir to halt sheath fluid flow or divert any remaining sheathfluid to the waste receptacle. Optionally, the flow analyzer includes asingle-filter light path from each optical detector to each amplifyingphoto-detector.

The diagnostic system, optionally, further includes a vertically and/orhorizontally moveable platform. The flow analyzer, optionally, includesa vertically moveable aspirator. The platform, optionally, cooperateswith the aspirator. The platform, optionally, supports a microtiterplate for the flow analyzer. The instruction for running the instantflow analyzer, optionally, includes exposing a pooled population ofsubsets of particles to the biological sample, the particles in eachsubset having (i) one or more classification parameters that distinguishthe particles of one subset from those of another subset, and (ii) areactant specific for each analyte of interest. The running instruction,optionally, further includes passing the exposed pooled population ofsubsets of particles through an examination zone.

The instruction for determining the identity and quantity of one or moreanalytes of interest in a biological sample, optionally, includesassessing the identify and quantity of each analyte of interest, ifpresent, in the sample by substantially contemporaneously performing thefollowing steps. Data is, optionally, collected relating to at least onecharacteristic classification parameter, including data on fluorescenceemission intensities. Data is, optionally, collected relating to apresence or absence of a complex formed between the reactant and ananalyte of interest specific to the reactant. Without relyingexclusively, if at all, on differences in particle size, each particleis classified according to its subset. An amount of complex associatedwith each subset is quantified. The step of collecting data relating toa presence or absence of a complex includes collecting analyte data onfluorescence emission intensities. The bead subset data and the analytedata optionally exhibit spectral overlap. The classifying step,optionally, includes reducing the spectral overlap sufficiently toidentify each bead according to its subset.

The diagnostic system, optionally, further includes a circular memorybuffer communicatable with the flow analyzer. The circular memory bufferoptionally includes a first movable pointer in operation, pointing to astorage position available for storing new data, and a second movablepointer, in operation, pointing to a storage position having unanalyzeddata.

It is also a feature and advantage of the instant invention to provide acuvette holder. The cuvette holder, optionally, includes a cuvetteholder top including one or more optional viewing grooves along one of adiameter and a width of the top. The cuvette holder, optionally, furtherincludes a cuvette holder base for cooperating with the top to hold acuvette. Optionally, the cuvette holder further includes a base frame.The cuvette holder top is secured to the base frame. The cuvette holderalso optionally includes a stability bracket secured to the base frameand securing a top of the cuvette.

It is another feature and advantage of the instant invention to providea computer program product. The computer program product, for example,includes a memory medium. The computer program product, for example,also includes a computer program stored on the memory medium. Thecomputer program, for example, contains sequential, non-sequential, orindependent instructions as follows. A biological sample is run througha flow analyzer. The biological sample includes a pooled population ofbead subsets. Each bead subset has one or more characteristicclassification parameters. The characteristic classification parameters,for example, includes one or more characteristic fluorescence emissionintensities. Substantially contemporaneously to the running step, datarelated to the at least one characteristic classification parameter, forexample, including bead subset data on fluorescence emission intensitiesis collected. Substantially contemporaneously to the running step, datarelated to the presence or absence of an analyte of interest, including,for example, analyte data on fluorescence emission intensities iscollected. The bead subset data and the analyte data optionally exhibitspectral overlap. Substantially contemporaneously to the running step,the spectral overlap is optionally reduced sufficiently to identify eachbead according to its subset.

The computer program optionally includes instructions for determining,substantially contemporaneously to the running step, a presence andquantity of one or more analytes of interest in the biological sample.

The computer program, optionally, further includes instructions forproviding a simplex analysis application module and/or a multiplexedanalysis application module. The computer program, optionally, furthercomprises or stores instructions for providing a main menu, a resultstable, a system monitor, a dot plot display including a density dot plotand/or a decaying dot ploy, a histogram tab, an optical amplifiercontrol tab, a color compensation control tab, and/or a doubletdiscriminator control tab.

It is also a feature and advantage of the instant invention to provide acomputer program product for use with a flow analyzer and a computer.The computer program product includes a memory medium and a computerprogram stored on the memory medium. The computer program contains thefollowing sequential, non-sequential, or independent instructions. Abiological sample is processed using, or run through, a flow analyzer.The biological sample includes a pooled population of bead subsets. Eachbead subset has one or more characteristic classification parameters.The one or more characteristic classification parameters includes one ormore characteristic fluorescence emission intensities. Substantiallycontemporaneously to the processing step, data, related to the at leastone characteristic classification parameter including bead subset dataon fluorescence emission intensities, is collected. Substantiallycontemporaneously to said processing step, data, related to a presenceor absence of an analyte of interest, including analyte data onfluorescence emission intensities, is collected. Substantiallycontemporaneously to the processing step, an identify and quantity of atleast one analyte of interest in the biological sample is determined.The computer program product also includes an application programminginterface library interfacing with the flow analyzer and the computerprogram, in operation. The computer program further includes amathematics library communicating with the computer program, inoperation.

The application programming interface library optionally includes one ormore of the following functions: a function for initializing a deviceinterface for the flow analyzer; a function for closing a device sessionwith a flow analyzer; a function for loading a map file fordistinguishing between the bead subsets; a function for defining beadsubsets to be associated with an assay; a function for acquiring beadstatistics of a selected bead subset; a function for copying flowanalyzer settings into a user-supplied buffer; and a function forchanging the flow analyzer settings.

The computer program product optionally includes one or more of thefollowing functions: a function for initiating acquisition of beadstatistics for a current sample loaded on the flow analyzer; a functionfor ending the acquisition of bead statistics; a function for copyingmost current bead statistics into a user-supplied buffer; and a functionfor one of returning and displaying data acquisition statistics.

It is another feature and advantage of the instant invention to providea multi-analyte diagnostic method having the following sequential,non-sequential, or independent steps. A biological sample is processedusing, or run through, a flow analyzer. The biological sample includes apooled population of bead subsets. Each bead subset has one or morecharacteristic classification parameters. The one or more characteristicclassification parameters includes one or more characteristicfluorescence emission intensities. Substantially contemporaneously tothe processing step, data, related to the at least one characteristicclassification parameter including bead subset data on fluorescenceemission intensities, is collected. Substantially contemporaneously tothe processing step, data related to a presence or absence of an analyteof interest, including analyte data on fluorescence emissionintensities, is collected. The bead subset data and the analyte dataexhibit spectral overlap. Substantially contemporaneously to theprocessing step, the spectral overlap is reduced sufficiently toidentify each bead according to its subset. Substantiallycontemporaneously to the processing step, an identify and quantity of atleast one analyte of interest in the biological sample is determined.

It is another feature and advantage of the instant invention to providea management system. The management system includes a file systemstoring static portions of substantially all data pages in a data site.The system also includes a server communicatably connected to the filesystem. The server retrieves the static portions of one or more datapages stored by the file system and transmits to a site user the staticportions of one or more data pages.

Optionally, the management system further includes a data page generatorgenerating the static portions of substantially all data pages based onthe data site for storage in the file system. The management systemfurther includes a dynamic data transmit device to transmit dynamic datato be cooperatively presented with the static portions as the at leastone data page to the site user. Optionally, the data page generatorgenerates the static portions of the substantially all data pages andprovides corresponding indexes therewith. Optionally, the servertransmits the static portions to the site user responsive to thecorresponding index associated with the at least one data page.

It is another feature and advantage of the instant invention to providea method of managing a data site having the following sequential,non-sequential, or independent steps. Static portions of substantiallyall data pages in a data site, are stored using a file system. Thestatic portions of at least one data page stored by the file system, areretrieved using a server communicatably connected to the file system.The static portions of the at least one data page, are transmitted to asite user using the server.

Optionally, the static portions of the substantially all data pagesbased on the data site for storage in the file system, are generatedusing a data page generator. Optionally, dynamic data to becooperatively presented with the static portions as the at least onedata page to the site user, are transmitted using a dynamic datatransmit device. Optionally, the data page generator generates thestatic portions of the substantially all data pages and providescorresponding indexes therewith. Optionally, the server transmits thestatic portions to the site user responsive to the corresponding indexassociated with the at least one data page.

It is another feature and advantage of the instant invention to providean analysis or diagnostic method, having the following sequential,sequence independent, or non-sequential steps. A plurality of pooledsubsets are processed through an inspection area, each of the pluralityof pooled subsets including one or more indication parameters. Each ofthe plurality of samples are illuminated, substantially simultaneouslyand not sequentially, with two or more light beams from one or moresources, at substantially the same time. One or more indicationparameters are determined responsive to the illuminating step.Optionally, each of the light beams according to this method includescontinuous wave light.

It is another feature and advantage of the instant invention to includea novel flow cytometer including a base section. A plurality of lightsources is mounted to the base section. A plurality of selectors ismounted to the base section. A sample viewing chamber is mounted to thebase section and in optical relationship with the plurality of lightsources and the plurality of detectors.

It is another feature and advantage of the instant invention to includean analysis or diagnostic system. The instant diagnostic systemaccording to this embodiment includes an initialization systeminitializing a device interface for a flow cytometer, including atermination system terminating a device session for the flow cytometer;a bead map file system loading a file defining a bead map indicative ofan associated bead type; a reset system resetting beads to be used inthe analysis or diagnostic system; and a user bead component system usedto acquire bead statistics for the beads and the associated bead type.

The instant diagnostic system according to this embodiment furtherincludes a machine control and monitoring system, responsively coupledto the initialization system, and monitoring and controlling theanalysis or diagnostic system, including: a panel setting systemmaintaining current flow cytometer settings in a buffer or storage area;and a change panel setting system changing at least one of the currentflow cytometer settings responsive to a command.

The instant diagnostic system according to this embodiment also includesa sample acquisition and reporting system, responsively coupled to themachine control and monitoring system, collecting data for analysis ordiagnosis, including: a test start system indicating when to begincollecting the data from the machine control and monitoring system; atest stop system indicating when to stop collecting the data from saidmachine control and monitoring system; a test stop system indicatingwhen to stop collecting the data from said machine control andmonitoring system; a test storage system storing the data in anotherbuffer or storage area; and a test query system performing the analysisor diagnosis on the data responsive to a predetermined program or userquery.

It is another feature and advantage of the instant invention to includean analysis or diagnostic method having the following independent,sequential, or non-sequential steps. A device interface for a flowcytometer is initialized. The initializing step includes terminating adevice session for the flow cytometer; loading a bead map file defininga bead map indicative of an associated bead type; resetting beads to beused in the analysis or diagnostic system; and acquiring bead statisticsfor the beads and the associated bead type.

An analysis or diagnosis is controlled and monitored using a machinecontrol and monitoring system. The controlling and monitoring stepincludes: maintaining current flow cytometer settings in a buffer orstorage area; and changing at least one of the current flow cytometersettings responsive to a command.

Data is collected for analysis or diagnosis. The data collection stepincludes: indicating when to begin collecting the data from the machinecontrol and monitoring system; indicating when to stop collecting thedata from the machine control and monitoring system; storing the data inanother buffer or storage area; and performing the analysis or diagnosison the data responsive to a predetermined program or user query.

It is another feature and advantage of the instant invention to providea detector apparatus including a U-block assembly. The detectorapparatus includes one or more optical beam splitters and one or moreoptical detectors secured to the U-block assembly. The detectorapparatus further includes one or more push-pull assemblies adjustablysecuring the one or more optical beam splitters to the U-block assembly,and directing the one or more optical beam splitters to be sufficientlyoptically couplable with the one or more optical detectors.

Optionally, the U-block assembly includes a unitary or integrated bodyhaving an inner portion. The one or more optical beam splitter bordered,in part, by the inner portion of the U-block assembly. The body of theU-block assembly includes first and second legs. The one or more opticaldetectors are secured within the first leg, and the one or morepush-pull assemblies are secured within the second leg. Optionally, thedetector apparatus further includes an optical assembly base frame,wherein the U-block assembly is secured thereto. The one or morepush-pull assemblies includes a screw tap and spring assembly forpushing a side of the one or more beam splitters toward the one or moreoptical detectors, and/or pulling another side of the one or more beamsplitters away from the one or more optical detectors.

Optionally, the one or more optical beam splitters includes one-or moredichroic mirrors. The one or more dichroic mirrors includes a pluralityof dichroic mirrors. Each dichroic mirror directs a respective band ofwavelengths of light to a respective optical detector and transmitting aremainder of wavelengths of light therethrough. Optionally, the detectorapparatus further comprises one or more filters, interposed between theone or more optical beam splitters and the one or more opticaldetectors.

It is also a feature and advantage of the instant invention to provide,in a flow analyzer including a pressure sensor and a sheath fluidreservoir, a de-bubbler. The novel de-bubbler includes a bottleincluding an upper portion and a lower portion. The upper portion of thebottle includes an inlet operatively connected to the sheath fluidreservoir to receive sheath fluid therefrom. The lower portion of thebottle includes an outlet operatively connected to the pressure sensor.The bottle further includes a substantially waterproof vent sealing atop of the bottle and exposing an interior of the bottle to anatmosphere external to the bottle so that, in operation, sheath fluid isoutput via the outlet substantially free of a gas bubble.

Optionally, the flow analyzer further includes a sample pump. Thepressure sensor optionally transmits a command to deactivate the samplepump upon sensing a decreased fluid pressure in the de-bubbler.

It is another feature and advantage of the instant invention to providea multi-analyte diagnostic system for analyzing a sample fluid for oneor more analytes of interest. The multi-analyte diagnostic systemincludes a flow analyzer, which includes a cuvette including, inoperation, a fluid core, and including a neck region having asubstantially flat glass-to-fluid interface and a substantially flatair-to-glass interface. The flow analyzer also includes a firstmagnification lens optically cooperative with the cuvette and having amagnification power. The flow analyzer further includes a filter andoptical amplifier assembly including an entrance aperture. The entranceaperture is dimensioned to cooperate with the magnification power totransmit light from the fluid core in the cuvette with substantially nolight distortion from the glass-to-fluid interface and/or theair-to-glass interface.

Optionally, the flow analyzer is communicatable with a computer. Themulti-analyte diagnostic system optionally includes a memory mediumreadable by the computer and storing computer instructions executed bythe computer. The computer instructions include processing the samplefluid using said flow analyzer, and analyzing the sample fluid anddetermining a presence and quantity of at least one analyte of interestin the sample fluid substantially simultaneously to the processing step.

Optionally the multi-analyte diagnostic system further includes a firstmirror optically coupled to the first magnification lens and reflectinga first plurality of wavelengths of the light to the entrance aperture,at least one of the first plurality of wavelengths indicative of apresence of one or more analytes of interest in the sample fluid.

Optionally, the flow analyzer further includes one or more light sourcesto radiate the cuvette. The one or more light sources include a laserdiode and/or a diode pumped laser.

Optionally, the cuvette includes upper and lower portions. Optionally,the multi-analyte diagnostic system further includes an optical assemblybase frame. The first magnification lens, the filter and opticalamplifier assembly, and the one or more light sources are secured to theoptical assembly base frame. The multi-analyte diagnostic system furtherincludes a cuvette holder secured to the optical assembly base frame andsecuring a bottom of the cuvette. The diagnostic system also includes anoptional stability bracket secured to the optical assembly base frameand securing a top of the cuvette.

Optionally, the multi-analyte diagnostic system further includes asecond magnification lens optically cooperative with the cuvette. Thediagnostic system optionally includes one or more optical beam splittersoptically cooperative with the second magnification lens. The diagnosticsystem optionally includes one or more optical detectors identifying oneor more particles as belonging to a respective particle subset, andoptically cooperative with the one or more optical beam splitters.

Optionally, the multi-analyte diagnostic system further includes asecond mirror optically coupled to the second magnification lens and theone or more beam splitters. The second mirror reflects a secondplurality of wavelengths of light to the one or more optical beamsplitters. One or more of the second plurality of wavelengths areindicative of the identity of the one or more particles.

Optionally, the multi-analyte diagnostic system further includes a sidescatter optically cooperating with the one or more beam splitters andidentifying a doublet.

There has thus been outlined, rather broadly, the more importantfeatures of the invention in order that the detailed description thereofthat follows may be better understood, and in order that the presentcontribution to the art may be better appreciated. There are, of course,additional features of the invention that will be described hereinafterand which will form the subject matter of the claims appended hereto.

In this respect, before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notlimited in its application to the details of construction and to thearrangements of the components set forth in the following description orillustrated in the drawings. The invention is capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology, and terminologyemployed herein are for the purpose of description and should not beregarded as limiting.

As such, those skilled in the art will appreciate that the conception,upon which this disclosure is based, may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

Further, the purpose of the foregoing abstract is to enable the U.S.Patent and Trademark Office and the public generally, and especially thescientists, engineers and practitioners in the art who are not familiarwith patent or legal terms or phraseology, to determine quickly from acursory inspection the nature and essence of the technical disclosure ofthe application. The abstract is neither intended to define theinvention of the application, which is measured by the claims, nor is itintended to be limiting as to the scope of the invention in any way.

These together with other objects of the invention, along with thevarious features of novelty which characterize the invention, arepointed out with particularity in the claims annexed to and forming apart of this disclosure. For a better understanding of the invention,its operating advantages and the specific objects attained by its uses,reference should be had to the accompanying drawings and descriptivematter in which there is illustrated preferred embodiments of theinvention.

Notations and Nomenclature

The detailed descriptions which follow may be presented in terms ofprogram procedures executed on a computer or network of computers. Theseprocedural descriptions and representations are the means used by thoseskilled in the art to most effectively convey the substance of theirwork to others skilled in the art.

A procedure is here, and generally, conceived to be a self-consistentsequence of steps leading to a desired result. These steps are thoserequiring physical manipulations of physical quantities. Usually, thoughnot necessarily, these quantities take the form of electrical ormagnetic signals capable of being stored, transferred, combined,compared and otherwise manipulated. It proves convenient at times,principally for reasons of common usage, to refer to these signals asbits, values, elements, symbols, characters, terms, numbers, or thelike. It should be noted, however, that all of these and similar termsare to be associated with the appropriate physical quantities and aremerely convenient labels applied to these quantities.

Further, the manipulations performed are often referred to in terms,such as adding or comparing, which are commonly associated with mentaloperations performed by a human operator. No such capability of a humanoperator is necessary, or desirable in most cases, in any of theoperations described herein which form part of the present invention;the operations are machine operations. Useful machines for performingthe operation of the present invention include general purpose digitalcomputers or similar devices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a prior art flow cytometer;

FIG. 2 is a schematic of a prior art flow cytometer;

FIG. 3 is a schematic showing operation of a flow cytometer;

FIG. 4 is a general schematic of an illustrative embodiment of theinstant diagnostic system;

FIG. 5 is a detailed schematic of an illustrative embodiment of part ofthe present system;

FIG. 6 is a detailed schematic of an illustrative embodiment of part ofthe present system;

FIG. 7 is a schematic of an illustrative embodiment of a circular memorystructure;

FIG. 8 is a perspective view of an illustrative embodiment of an arrayof light sources and optical detectors in the instant flow analyzer;

FIG. 9 is a perspective view of an illustrative embodiment of thepresent light source-optical detector array;

FIG. 10 is a perspective view of an illustrative embodiment of theinstant flow analyzer;

FIG. 11 is a perspective view of an illustrative embodiment of thecircuitry of the instant flow analyzer;

FIG. 12 is a perspective view of an illustrative embodiment of a fluidbag and a vial;

FIG. 13 is a perspective view of an illustrative embodiment of thefluidics system in the instant flow analyzer;

FIG. 14 is a schematic of an illustrative embodiment of the instantfluidics system;

FIG. 15 a is a perspective view of an illustrative embodiment of acuvette holder;

FIG. 15 b is an exploded, perspective view of the cuvette holder shownin FIG. 15 a;

FIG. 16 is a perspective view of an illustrative embodiment of a cuvetteholder top;

FIG. 17 is a perspective view of an illustrative embodiment of a laserhousing;

FIG. 18 is a perspective view of an illustrative embodiment of adetector housing;

FIG. 19 is a perspective view of an illustrative embodiment of a movableplate transport cooperating with a flow analyzer;

FIG. 20 is an illustrative embodiment of an initial screen display ofthe instant control and analysis software;

FIG. 21 is an illustrative embodiment of a grid having predeterminedbead regions of characteristic fluorescence emissions;

FIG. 22 is an illustrative embodiment of an overall screen display;

FIG. 23 is an illustrative embodiment of a title bar and main menu bar;

FIG. 24 is an illustrative embodiment of a new folder graphical displaywindow;

FIG. 25 is an illustrative embodiment of a machine setup graphicaldisplay window;

FIG. 26 is an illustrative embodiment of a results table;

FIG. 27 is an illustrative embodiment of an assay information graphicaldisplay window;

FIG. 28 is an illustrative embodiment of an assay information graphicaldisplay window;

FIG. 29 is an illustrative embodiment of a graphical display of a systemmonitor;

FIG. 30 is an illustrative embodiment of a graphical display of ahistogram tab graphical display;

FIG. 31 is an illustrative embodiment of an optical amplifiers tabgraphical display;

FIG. 32 is an illustrative embodiment of a color compensation tabgraphical display;

FIG. 33 is an illustrative embodiment of a doublet discriminator tabgraphical display;

FIG. 34 is an illustrative embodiment of a dot plot graphical display;

FIG. 35 is an illustrative embodiment of a density plot graphicaldisplay window;

FIG. 36 is an illustrative embodiment of an assay information graphicaldisplay window;

FIG. 37 is an illustrative embodiment of a bead details graphicaldisplay window;

FIG. 38 is an illustrative embodiment of a computer and assortedperipherals;

FIG. 39 is an illustrative embodiment of computer architectureconsistent with the instant invention;

FIG. 40 is an illustrative embodiment of a memory medium;

FIG. 41 is a flow chart of an illustrative embodiment of a method ofoperation for the instant diagnostic system;

FIG. 42 is a flow chart of an illustrative embodiment of a method ofoperation for a flow analyzer consistent with the instant invention;

FIG. 43 is a schematic of an embodiment of the computer-implementedprocess cooperating with the flow analyzer;

FIG. 44 is a block diagram of exemplary components of an applicationprogramming interface library and of a control and analysis softwareconsistent with the instant invention;

FIG. 45 is a schematic of an alternative embodiment of the instantinvention;

FIG. 46 is a perspective view of a modified cuvette;

FIG. 47 is a perspective view of exemplary optical assembly componentsof the alternative embodiment;

FIG. 48 is a planar view of exemplary optical assembly components of thealternative embodiment;

FIG. 49 is a perspective view of a de-bubbler according to the instantinvention;

FIG. 50 is a perspective view of an exemplary optical switch accordingto the instant invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Generally, the instant multi-analyte diagnostic system performs on abiological sample, bioassays including, for example, immunoassays,complex genetic analyses, and enzymatic assays. To this extent andothers U.S. application Ser. No. 08/540,814 to Van S. Chandler et al. isincorporated herein by reference in its entirety.

The biological sample to be tested using the instant invention, forexample, includes plasma, serum, tears, mucus, saliva, urine, pleuralfluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretion,fluid from ulcers and/or other surface eruptions, blisters, abscesses,and/or extracts of tissues, such as biopsies of normal, malignant,and/or suspect tissues.

The analytes of interest for these bioassays include, for example,antigens, antibodies, autoantibodies, peptides, proteins, nucleic acidsequences, and/or enzymes. The antigenic analytes, for example, includesbacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial, and/orprotozoal antigens. Alternatively, the antigens, for example, includeantigens borne by pathogenic agents responsible for a sexuallytransmitted disease, antigens borne by pathogenic agents responsible fora pulmonary disorder, and/or antigens borne by pathogenic agentsresponsible for gastrointestinal disorder.

The analyte of interest, for example, includes a substance of abuse or atherapeutic drug. The analyte of interest, for example, includes anantigen or antibody associated with a pathological syndrome, such ascardiovascular disorders, malignancy, allergy, autoimmune diseases,and/or blood-borne viruses. The analyte, for example, is an indicatorfor pregnancy or specific hormones.

The enzymatic analytes includes, for example, proteases, glycosidases,nucleotidases, oxidoreductases, hydrolases, esterases, convertases,ligases, transferases, phosphorylases, lyases, lipases, peptidases,dehydrogenases, oxidases, phospholipases, invertases, aldolases,transaminases, synthetases, and/or phosphotases.

As shown in FIG. 4, the system components of the instant inventioninclude a flow analyzer 25, such as a flow cytometer, and a cooperativecontrol and analysis software package 50. Hardware components for usewith the instant invention, for example, include a power source,interface cable, and/or a standard computer. Consumables for use of theinstant invention, for example, include microliter tubes, for example,of 1 mL each, sheath fluid, and microspheres. Optional components, forexample, include, for example, a standard spreadsheet software packagecooperatively linked to the control and analysis software of the instantinvention.

The microspheres are alternately termed microparticles, beads,polystyrene beads, microbeads, latex particles, latex beads, fluorescentbeads, fluorescent particles, colored particles and colored beads. Themicrospheres serve as vehicles for molecular reactions. Microspheres foruse in flow cytometry are obtained from manufacturers, such as LuminexCorp. of Austin, Tex. Illustrative microspheres and methods ofmanufacturing same are, for example, found in U.S. patent applicationSer. No. 09/234,841 to Mark B. Chandler and Don J. Chandler, entitledMicroparticles with Multiple Fluorescent Signals, and in U.S. patentapplication Ser. No. 09/172,174 to Don J. Chandler, Van S. Chandler, andBeth Lambert, entitled Precision Fluorescently Dyed Particles andMethods of Making and Using Same, both patent applications incorporatedherein by reference in their entirety. By way of example, if a user wereperforming an Ig G, A, M Isotyping Assay, the user opts for bead sets,such as Luminex 8070 IgG, 8060 IgA, and 8050 IgM bead sets.

Preferably, the microspheres used according to the instant inventioninclude a pooled population of classes or subsets of microspheres. Eachsubset of microspheres includes one, two, or more microspheres.Advantageously, a plurality of microspheres per subset are used, forexample, up to 1000 or more. Each microsphere in a respective subset ofmicrospheres includes one, two, three, four, five or more classificationparameters. For example, one classification parameter includes a forwardlight scatter parameter, and another includes a side light scatterparameter.

The classification parameters of each microsphere advantageouslyincludes one, two, three, or more standard fluorochromes or fluorescentdyes. The one or more fluorochromes are affixed to or embedded in eachmicrosphere by any standard method, for example, by attachment to themicrosphere surface by covalent bonding or adsorption. Alternatively,the dye(s) may be affixed by a copolymerization process, whereinmonomers, such as an unsaturated aldehyde or acrylate, are allowed topolymerize in the presence of a fluorescent dye, such as fluorosceinisothiocynate (FITC), in the resulting reaction mixture.

Another method by which one or more dyes are embedded in a microsphereincludes adding a subset of microspheres to, for example, an organicsolvent to expand the microspheres. An oil-soluble or hydrophobic dye,for example, is subsequently added to the subset of microspheres,thereby penetrating into each microsphere. After incubating theresulting combination, an alcohol or water-based solution, for example,is added to the combination and the organic solvent is removed. Themicrosphere shrinks, retaining the dye(s) inside. Each fluorochrome inthe microsphere optionally serves as an additional or alternativeclassification parameter.

The microsphere classes include respective reporter substances such asantibodies, antigens, peptides, proteins, enzymes and/or nucleic acidprobes to provide specific signals for each reaction in a multiplexedassay. Each reporter substance is selected to react, optionallyuniquely, to an analyte of interest in a biological sample.

FIG. 39 is an illustration of a main central processing unit forimplementing the computer processing in accordance with a computerimplemented embodiment of the present invention. The proceduresdescribed herein are presented in terms of program procedures executedon, for example, a computer or network of computers.

Viewed externally in FIG. 39, a computer system designated by referencenumeral 900 has a computer 902 having disk drives 904 and 906. Diskdrive indications 904 and 906 are merely symbolic of a number of diskdrives which might be accommodated by the computer system. Typically,these would include a floppy disk drive 904, a hard disk drive (notshown externally) and a CD ROM indicated by slot 906. The number andtype of drives varies, typically with different computer configurations.Disk drives 904 and 906 are in fact optional, and for spaceconsiderations, are easily omitted from the computer system used inconjunction with the production process/apparatus described herein.

The computer system also has an optional display 908 upon whichinformation is displayed. In some situations, a keyboard 910 and a mouse902 are provided as input devices to interface with the centralprocessing unit 902. Then again, for enhanced portability, the keyboard910 is either a limited function keyboard or omitted in its entirety. Inaddition, mouse 912 optionally is a touch pad control device, or a trackball device, or even omitted in its entirety as well. In addition, thecomputer system also optionally includes at least one infraredtransmitter and/or infrared received for either transmitting and/orreceiving infrared signals, as described below.

FIG. 39 illustrates a block diagram of the internal hardware of thecomputer system 900 of FIG. 38. A bus 914 serves as the main informationhighway interconnecting the other components of the computer system 900.CPU 916 is the central processing unit of the system, performingcalculations and logic operations required to execute a program. Readonly memory (ROM) 918 and random access memory (RAM) 920 constitute themain memory of the computer. Disk controller 922 interfaces one or moredisk drives to the system bus 914. These disk drives are, for example,floppy disk drives such as 904, or CD ROM or DVD (digital video disks)drive such as 906, or internal or external hard drives 924. As indicatedpreviously, these various disk drives and disk controllers are optionaldevices.

A display interface 926 interfaces display 908 and permits informationfrom the bus 914 to be displayed on the display 908. Again as indicated,display 908 is also an optional accessory. For example, display 908could be substituted or omitted. Communications with external devices,for example, the components of the apparatus described herein, occursutilizing communication port 928. For example, optical fibers and/orelectrical cables and/or conductors and/or optical communication (e.g.,infrared, and the like) and/or wireless communication (e.g., radiofrequency (RF), and the like) can be used as the transport mediumbetween the external devices and communication port 928. Peripheralinterface 930 interfaces the keyboard 910 and the mouse 912, permittinginput data to be transmitted to the bus 914.

In addition to the standard components of the computer, the computeralso optionally includes an infrared transmitter and/or infraredreceiver. Infrared transmitters are optionally utilized when thecomputer system is used in conjunction with one or more of theprocessing components/stations that transmits/receives data via infraredsignal transmission. Instead of utilizing an infrared transmitter orinfrared receiver, the computer system optionally uses a low power radiotransmitter and/or a low power radio receiver. The low power radiotransmitter transmits the signal for reception by components of theproduction process, and receives signals from the components via the lowpower radio receiver. The low power radio transmitter and/or receiverare standard devices in industry.

FIG. 40 is an illustration of an exemplary memory medium 932 which canbe used with disk drives illustrated in FIGS. 38 and 39. Typically,memory media such as floppy disks, or a CD ROM, or a digital video diskwill contain, for example, a multi-byte locale for a single bytelanguage and the program information for controlling the computer toenable the computer to perform the functions described herein.Alternatively, ROM 918 and/or RAM 920 illustrated in FIGS. 38 and 39 canalso be used to store the program information that is used to instructthe central processing unit 916 to perform the operations associatedwith the production process.

Although computer system 900 is illustrated having a single processor, asingle hard disk drive and a single local memory, the system 900 isoptionally suitably equipped with any multitude or combination ofprocessors or storage devices. Computer system 900 is, in point of fact,able to be replaced by, or combined with, any suitable processing systemoperative in accordance with the principles of the present invention,including sophisticated calculators, and hand-held, laptop/notebook,mini, mainframe and super computers, as well as processing systemnetwork combinations of the same.

Conventional processing system architecture is more fully discussed inComputer Orqanization and Architecture, by William Stallings, MacMillanPublishing Co. (3rd ed. 1993); conventional processing system networkdesign is more fully discussed in Data Network Design, by Darren L.Spohn, McGraw-Hill, Inc. (1993), and conventional data communications ismore fully discussed in Data Communications Principles, by R. D. Gitlin,J. F. Hayes and S. B. Weinstain, Plenum Press (1992) and in The IrwinHandbook of Telecommunications, by James Harry Green, Irwin ProfessionalPublishing (2nd ed. 1992). Each of the foregoing publications isincorporated herein by reference. Alternatively, the hardwareconfiguration is, for example, arranged according to the multipleinstruction multiple data (MIMD) multiprocessor format for additionalcomputing efficiency. The details of this form of computer architectureare disclosed in greater detail in, for example, U.S. Pat. No.5,163,131; Boxer, A., Where Buses Cannot Go, IEEE Spectrum, February1995, pp. 41-45; and Barroso, L. A. et al., RPM: A Rapid PrototypingEngine for Multiprocessor Systems, IEEE Computer February 1995, pp.26-34, all of which are incorporated herein by reference.

In alternate preferred embodiments, the above-identified processor, and,in particular, CPU 916, may be replaced by or combined with any othersuitable processing circuits, including programmable logic devices, suchas PALs (programmable array logic) and PLAs (programmable logic arrays).DSPs (digital signal processors), FPGAs (field programmable gatearrays), ASICs (application specific integrated circuits), VLSIs (verylarge scale integrated circuits) or the like.

By way of illustration, the computer 900 includes a personal computer,such as, a Pentium MMX 166 microprocessor-powered personal computer, asmanufactured, for example, by Dell Computer Corporation of Round Rock,Tex., and having, for example, 32 megabytes of RAM, 2 megabytes of VRAM,a 2.0 gigabyte hard drive, a keyboard, a mouse, a 1024×768 resolutionSVGA color monitor, a CD-ROM, and/or a digital signal processor. Thepersonal computer, for example, is network capable. The computer 900runs the control and analysis software including initialization,calibrations set-up, data acquisition, filtering, statistics, resultscalculation, and report printing.

It is, of course, understood that the computer is alternatively embodiedas a thin client, such as a network computer or a NetPC, incommunication with a flow analyzer, wherein control and analysissoftware, discussed below, resides on a network accessible by the thinclient. Alternatively, the computer is embodied as a minicomputer or amainframe in communication with a flow analyzer, wherein resides thecontrol and analysis software.

As shown in FIGS. 4-6, the personal computer 90 is operatively connectedto a flow analyzer, for example, via a standard serial or parallelconnection interface card 15, which provides a communications interfacebetween the computer bus of the computer and a flow analyzer data bus 20of the flow analyzer 25.

The software 50 in the computer communicates with the flow analyzer 25through the serial or parallel connection interface card 15 as follows.The flow analyzer 25 updates the communications interface with a blockof parameters describing the flow analyzer's current state. Pertinentinformation includes, for example, the state of one or more of itsphotomultiplier tubes, fluid levels, etc. Using the serial or parallelconnection interface card 15, the software optionally reports thecurrent status of these parameters to the user. For example, thesoftware 50 warns the user if, for example, the sheath fluid containeris empty and/or requires refilling. The software 50 optionally alsowarns the user if, for example, the waste fluid container is full and/orrequires emptying. The software 50 optionally further notifies the userif, for example, a pressure-related problem exists. An error message,such as “Bad Link,” is optionally displayed, if, for example, it isdetermined that the flow analyzer 25 and the computer 10 are notcommunicating properly. The software 50 optionally monitors and comparesthe flow analyzer's settings to ensure that they fall within acceptablecalibrated setting guidelines or settings. The software 50 optionallyreports a parameter change to the user promptly after receiving thechange via the serial or parallel connection interface card 15.

The flow analyzer 25, for example, reports light scatter events in thesample that passes therethrough and, for example, are detected above athreshold value, that is pre-set or user-defined. The flow analyzer 25sends the events across the serial or parallel connection interface card15 in, for example, a standard list mode data format, which facilitatesdata export to standard third-party programs and spreadsheets.Advantageously, such a format aids in systems integration of elementsproduced by disparate manufacturer's to create the instant invention. Byway of illustration, events are optionally sent in blocks of anarbitrary fixed number, such as fifteen, or a variable number. Eachevent contains the detected amount of light at each photomultipliertube, for example, and, optionally, a checksum to ensure propertransmission. Each event from a photomultiplier, for example, asrepresented by the amount of detected light is received as, for example,a linear value of a number of bits in length. The number of bits is, forexample, one, two, three, ten, twenty, or more. Plainly, an event beingdescribed by a number of bits lower than ten is coarser than one beingdescribed by ten or more bits. By increasing or decreasing the number ofbits used in the event value parameter, the resolution of the event, andthus the dynamic range thereof, is increased or decreased, respectively.Optionally, the software 50 in the computer 10 discards events thatproduce the checksum errors.

As shown in FIG. 6, the digital interface board 15 includes a standardmicrocontroller 60, such as the C167 microcontroller manufactured bySiemens AG of Munchen, Germany, and a standard digital signal processor(DSP) 65, such as the ADSP-2181 processor manufactured by AnalogDevices, Inc. of Norwood, Mass. Advantageously, the digital interfaceboard 15 is optionally protected from radiated noise by a standardshield. In addition, an optional heatsink covers the amplifyingphoto-detectors, such as, avalanche photo-diodes, so as to equalizetemperature while providing a secure mount for the light collimationoptics and the optical fibers.

The digital interface board 15, for example, measures collected lightfrom up to three or more channels and may, for example, resolve totallight emitted for every event. In flow analysis, a light scatter event,for example, includes an instance of a cell or other particle passingthrough a spot or focal region of light, such as a laser, and scatteringboth the excitation light and, often, one or more fluorescent colorsemitted from dye in the particle or cell. As the event occurs, themeasured light increases as the particle enters the beam. The high speedanalog-to-digital converters on the digital interface board 15,according to the instant invention, optionally continually orintermittently measures the light. Every channel is optionally sampledor measured, for example, every millionth of a second. Channels, ofcourse, are optionally measured at rates greater than or less than everymillionth of a second.

These measurements are optionally stored sequentially in an optionalstandard circular memory or standard buffer 30 in the interface card 15,as shown schematically in FIG. 7. A first pointer indicates the oldeststorage position 40 in the memory, which is the position where the nextmeasured channel sample will be stored. The circular buffer 30 has, forexample, 1,000 storage positions for measurements from each channel.Note the number of storage positions depicted in FIG. 4 is solelyintended for drawing convenience and is in no way intended to belimiting the scope of the invention. The circular buffer optionallyincludes greater than 1,000 storage positions or less than 1,000 storagepositions, as the speed of the flow analyzer 25 sampling warrants. Then,for example, if the flow analyzer 25 according to the instant inventionsamples at 1,000,000 samples per second and the buffer includes 1,000storage positions, the circular buffer 30 optionally holds measurementsfor analog-to-digital conversion from the previous 1/1000 of a second.

The DSP 65 on the digital interface board 15 optionally controls asecond pointer 45 for the circular buffer 30. The second pointeroptionally is programmed to stay a fixed trailing distance in timebehind the first pointer 35 until the measured light exceeds a thresholdvalue, e.g., a desired signal to noise ratio. It then moves forward,processing the measurements and analyzing the pulse. There are a numberof processes known to those of ordinary skill in the art that areoptionally performed on the pulse measurements, including digitalfiltering and/or waveform analysis, for example, that improve thequality of the measurement by extracting a signal in the presence ofnoise. By way of illustration, standard FIR filtering is advantageouslyused to improve the signal-to-noise ratio and, therefore, sensitivity.

At the conclusion of this signal processing, the DSP 65 advances itssecond pointer 45 until a new event is detected or until the secondpointer reaches the fixed trailing distance.

The process optionally continues until all or substantially all eventshave been measured. It is important to note that digital filtering maytake considerable time. The circular buffer size advantageously isoptionally large enough to handle the worst case scenario for maximumdata storage to adequately accommodate processing time. A fast flowcytometer 25 handles, for example, 20,000 cells or beads per second. Inthe preferred embodiment, wherein, for example, 1,000 events areprocessed per second, that rate results in an average of only twentymeaningful measurements of a given event in the circular buffer 30.Thus, for example, a buffer 30 having 1,000 storage positions would farexceed this data storage need.

The interface data bus cable 20 provides a conduit for setup parametersto the flow analyzer 25 from the computer 90 via the serial and parallelconnection 15. The cable 20 also provides a conduit for flow analyzerdata output from the flow analyzer 25 to the computer 90 via the serialor parallel connection interface card 15.

The computer 90 is optionally also operatively connected to an optionalprinter 55 for printing one or more reports generated by the control andanalysis software of the instant invention. The printer interfaces withthe computer via a standard printer port, such as a Centronics printerport manufactured by Genicon Corporation of Waynesboro, Va.

Flow Analyzer Overview

As illustrated in FIGS. 8-13, flow analyzer 25 includes, for example, astandard flow cytometer capable of analyzing cells or particles by sizeand/or by fluorescence, distinguishing up to three, for example, or morefluorescent colors simultaneously. Optionally, the flow cytometer is abench-top type model. The instant flow analyzer 25 is operativelyconnected to the personal computer 910 via a communications port. Ingeneral, the flow cytometer 25 advantageously integrates lasers, optics,fluidics, and advanced signal processing and/or have a small, forexample, 17″×20″ footprint. Such a flow cytometer 25, for example,includes a largely solid-state device.

More specifically, the flow cytometer 25 includes an examination zone70, for example, a sample delivery viewing chamber or flow cell. Theviewing chamber 70 is a standard quartz cuvette used in standard flowcytometers. Optionally, the cuvette 70 includes one or more flatair-to-glass interfaces. For example, as shown schematically in FIG. 14,the cuvette 70 has a hexagonal cross-section, thus effectively havingsix flat air-to-glass interfaces. Optionally, the number of air-to-glassinterfaces of the cuvette equals the number of light sources anddetectors.

As illustrated in FIGS. 15 and 16, the flow analyzer 25 includes anoptional cuvette holder top 190 which, for example, connects to a topplate or laser base of the light sources. The cuvette holder top 190includes an optional viewing groove or grooves along a diameter or widthof the holder top. The flow analyzer 25 includes a cuvette holder base,which optionally comprises two units 200 a, 200 b, wherein the cuvette70 is held between the cuvette holder base 200 a, 200 b and the cuvetteholder top 190.

As shown in FIG. 14, the cuvette is operatively connected to a sheathfluid reservoir or bag 75 and a sample fluid container 80. A standardcompressor pump 165, for example, pumps air into the reservoir 75,forcing sheath fluid to flow from the sheath fluid reservoir 75 througha standard filter 85 and a standard flow arrestor 90, which isoperatively connected to a standard sheath fluid pressure sensor 95, tothe cuvette. The flow arrestor 90, for example, includes a pinch valveor a solenoid valve. The sample container 80, is connected to thecuvette 70 via, for example, a standard syringe pump 100 in such amanner as to ensure that the sheath fluid from the sheath fluid bag 75ensheathes the sample fluid 170 before or in the cuvette 70.

Optionally, the exit end of the cuvette 70 is operatively connected viaa standard check valve 105 to a waste fluid bag 110. Optionally, thesyringe pump 100 for the sample fluid is also connected by a check valve115 to the same waste fluid bag 110 or a different waste fluid bag.

On an optional top plate or laser base 180, standard light sources 120and standard optical detectors 123 optionally surrounds the periphery ofthe viewing chamber 70, as configured, by way of example, in FIG. 9. Thelight sources 120, for example, includes standard laser diodes orstandard light emitting diodes. For example, dual diode lasers areoptionally horizontally opposed and are optionally horizontally pitcheddown approximately five degrees. A pitch having a fewer number or agreater number of degrees is also acceptable. By way of furtherillustration, one or both of the light emitting diodes optionallyincludes continuous wave (CW) light emitting diodes emitting one, two,or more wavelengths or wavelength bands of light. All of the lightsources 120 share identical, substantially identical, similar, oroverlapping focal regions in the viewing chamber 70 on the flowingsample fluid 170, in operation. The focal region includes across-section of, for example, 60 microns×30 microns, although othergeometries and sizes are also applicable to the present invention.

The optional top plate or laser base 180 is, for example, a unitaryplate or a rigidly connected plurality of plate for providing stabilityto the optical assembly. Such stability, for example, facilitates themaintenance of the above-mentioned identical, similar, or overlappingfocal regions. Advantageously, the rigid top plate 180 reduces thefrequency for any recalibration of the focal regions of the lightsources.

Optionally, one or more light sources are optionally located in ahousing, such as laser housing 175, which advantageously enhance therigidity of the light source detector assembly, as shown in FIG. 17.Each laser housing is optionally and advantageously affixed to the topplate 180. Similarly, optionally, one or more optical detectors islocated in a detector housing 185, which is also or alternatively,affixed to the top plate 180, as shown in FIG. 18.

The above-mentioned light sources, alternatively or in addition,include, by way of example, laser diodes, broad spectrum arc lamps,including mercury and xenon arc lamps, standard flash lamps, as well aslasers, including He—Ne, Ar-ion, Ar/Kr, UV, and YAG lasers, and othersuitable standard light sources.

Lasers that can emit light at more than one wavelength simultaneously orsubstantially simultaneously can also be used. For example, in thisregard, there is at least one laser which emits at 488 nm and 357 nmsimultaneously. In this situation, one laser would replace two singlebeam lasers/light sources. Alternatively, frequency tunable lasers arealso acceptable. Frequency tunable lasers include, for example, dyelasers, excimer lasers, and semiconductor lasers. Use of a frequencytunable laser optionally reduces the number of required light sources.Likewise, frequency tunable laser diodes and other frequency tunablelight sources are also within the scope of the invention.

The arc lamps optionally require conventional polarizers in the lightpath toward the viewing chamber. The lasers require standard beamshaping prismatic expanders or their equivalents between the lightsources and the flow cell. A possible disadvantage to using arc lampsand lasers as well as polarizers and prismatic expanders, is increasedsize of the overall apparatus relative to an embodiment using laserdiodes. Also, the lasers that emit at more than one wavelength oftenrequire different prismatic expanders for each wavelength. But, forpractical reasons, one set of prismatic expanders are, for example, usedfor both wavelengths.

It is to be understood that references made hereinafter to lasers and/orlight sources, unless otherwise specified, are made by way ofconvenience and are not intended to exclude the above-mentionedacceptable light sources.

The standard optical detectors 125 are connected via, for example, atleast one standard multi-mode fiber optic cable 130 to seriallyconnected amplification, filtering, and digital conversion units. Theamplification units optionally include standard optical amplifiers 135,for example, one or more standard avalanche photodiodes. The multi-modefiber optic cable 130 includes, for example, at least as many bundles asamplification units, each bundle operatively connected to a respectivestandard amplification unit. Each amplification unit is operativelyconnected to a respective filtering unit. Each filtering unit includesone or more band-pass filters 155, each having a band-pass frequency orfrequency bands corresponding to known emission wavelengths orwavelength bands. Optionally, each bundle in the multi-mode fiber opticcable is connected to a standard single amplification, filtering, anddigital conversion unit via a multiplexer or selector. Such animplementation, for example, trades off processing time formanufacturing complexity.

The number of optical detectors 125, optionally, depend at least in parton the total number of wavelengths emitted by the light sources 120.That is, for example, if there are two light emitting diodes, eachemitting two, respective, distinct wavelengths of light, four opticaldetectors are optionally implemented, one for each emitted wavelength oflight.

Alternatively, the number of optical detectors 125 may not be socorrelated. That is, for example, if there are two light emittingdiodes, each emitting two respective, distinct wavelengths of light,then, for example, one optical detector, connected to a multi-mode fiberoptic cable having four or more bundles, is optionally implemented.Alternatively, if there are two light emitting diodes, each emittingtwo, respective, distinct wavelengths of light, for example, two opticaldetectors, each having a multi-mode fiber optic cable including twobundles, is optionally implemented.

Optionally, if multiple optical detectors are implemented, themulti-mode fiber optic cables therefrom are optionally connected to oneor more standard multi-pass filters. Alternatively, if a single opticaldetector is implemented, the multi-mode fiber optic cable therefrom areoptionally connected to one or more multi-pass filters. Use of amulti-pass filter optionally entails serially connected low pass filtersto isolate signals in desired frequency bands. Implementation of one ormore multi-pass filters and additional low-pass filters, for example,add to the size and complexity of the instant invention in comparison toimplementation of multiple band-pass filters.

As shown in FIG. 6, each band-pass filter 155 optionally is connected inseries with, for example, a standard avalanche photodiode 135, astandard inverting amplifier 140, a standard low-pass Nyquist filter145, and a standard analog-to-digital converter 150. Alternatively, oneor more of the avalanche photodiodes 135 is optionally replaced withoptical amplifiers or photo-detectors known in the industry, such asphotomultiplier tubes and optically amplified photodiodes. The cutofffrequency for the Nyquist filter 145, for example, is 450 kHz. It isunderstood that the cutoff frequency optionally includes another valueconsistent with the characteristics of the Nyquist filter 145 in theinstant configuration. The analog-to-digital converters 150, in turn,are optionally connected in parallel to standard digital signalprocessor 65. The DSP 65 optionally polls the parallel inputssequentially, substantially simultaneously or simultaneously.Alternatively, the analog-to-digital converters 150 are optionallycoupled or connected to a standard selector or standard multiplexer,which in turn is optionally connected to the digital signal processor65. The selector passes signals from a single analog-to-digitalconverter 150 to the digital signal processor 65. Such a selectoradvantageously permits implementation of a less sophisticated digitalsignal processor than might otherwise be needed to handle parallelinputs from multiple analog-to-digital converters.

Optionally, the instant invention optionally includes two or moredigital signal processors working in parallel or in sequence. In such acase, a selector, such as the serially configured multiplexer anddemultiplexer, selects which of the analog-to-digital converters cantransfer its data to which of the digital signal processors at a giventime. Parallel digital signal processors may increase the cost of theinstant invention, but may also increase the speed of data processingfor analyte determination.

An alternative embodiment of the instant flow analyzer is shown, by wayof example, in FIG. 45. Components identical to those in theabove-described embodiment are given identical reference numerals. Assuch, only the differing components or configurations of the alternativeembodiment will be described hereinbelow.

I have recognized that a curved or rounded inner interface of a cuvette,for example, the sheath fluid-to-glass interface of a quartz cuvette, isa contributor to light distortion or background noise levels. Such aninner interface impedes measurement of emitted fluorescence because thecurved interface effects a sub-optimal level of light scattering withinthe cuvette.

I have determined that optional modified cuvette 800 including a flatair-to-glass interface and a flat glass-to-fluid interface, as shown, byway of example, in FIG. 46, addresses this very shortcoming. Modifiedcuvette 800 includes a square or rectangular cross-section.Advantageously and unexpectedly, the modified cuvette 800 including aneck-up region having a flat air-to-glass interface, a flatglass-to-fluid interface, and a square cross-section improvessensitivity of the instant invention between ten and one hundred timesthan otherwise possible with a cuvette having a rounded glass-to-fluidinterface.

By way of illustration, the modified cuvette 800 optically cooperateswith one or more optional standard magnification lens 801, 802, as shownin FIGS. 47 and 48. Optionally, magnification power of the lenses 801,802 is between approximately 20× and 40×. Advantageously, themagnification power of lenses 801, 802 is 25×. The magnification lens801 magnifies light from the modified cuvette 800, directing the lightvia an entrance slit 808 to a filter and optical amplifier assembly 810.Advantageously, optional mirror 805 reflects light from lens 801 to theentrance slit 808. The mirror 805 optionally includes a dichroic mirrorreflecting light having wavelengths, for example, in a range of 550 nmand 610 nm. Use of the mirror 805 facilitates miniaturization of theflow analyzer.

The entrance slit 808 is sized sufficient to pass light relating to thefluid in the cuvette. Optionally, the size of the entrance slit 808 islimited so as to block light from the glass-to-fluid interface to theglass-to-air interface. For example, if the fluid core in a cuvette istwelve microns in cross-section, the slit 808 is between about twelvemicrons and about one tenth of an inch or 40 microns. Light from, forexample, a twelve micron fluid core falls well within a forty micronentrance slit 808, thereby permitting jiggling of the instant apparatuswith little or no degradation of performance.

Optionally, a dichroic mirror or reflector 843, as shown, by way ofexample, in FIG. 48, is placed in front of the entrance slit 808 toreflect light having wavelengths associated with bead identification.For example, the dichroic reflector 843 reflects light in a range of 630nm to 760 nm to eliminate wavelengths of light to be used by theinfrared wavelength detector, the red wavelength detector, and/or theside scatter detector, as described below.

Optionally, the filter and optical amplifier assembly 810 identifies apresence and/or quantity of one or more analytes of interest in a samplefluid. By way of illustration, the presence of an analyte of interest isidentified by one or more fluorescence emission intensities, such as anorange fluorescence emission having a 585 nm wavelength. By way ofexample, the filter and optical amplifier assembly 810 includes abandpass filter and a photomultiplier tube. Other suitable types ofoptical amplifiers, such as avalanche photodiodes, are acceptable. Thebandpass filter passes, for example, 565 to 585 nm light so that thephotomultiplier tube detects a presence of 585 nm light. Plainly, othercolors and/or wavelengths are acceptable for identifying an a presenceof an analyte of interest, provided that they are distinguishable fromany other fluorescence emissions excited during the course of operationof the instant invention. Advantageously, magnification lens 802magnifies light emitted from the cuvette 800 to one or more opticaldetectors, for example, for bead identification. For example, the one ormore detectors include three detectors for detecting one or morecharacteristic classification parameters of a bead. By way ofillustration, classification parameters optionally includecharacteristic fluorescence emission intensities and/or bead size. Forinstance, beads of a bead subset in a multiplexed analysis areoptionally distinguished from beads of another subset by dyes havingdiffering fluorescence emission intensities of red light, e.g., 658 nmlight, and infrared light, e.g., 712 nm light. In such a case, forexample, red wavelength detector 822 and an infrared wavelength detectorare advantageously implemented. By way of illustration, referring toFIG. 48, light passing through magnification lens 802 partially reflectsoff of a beam splitter through a standard bandpass filter 821 passing,for example, light having wavelengths in a range of 700 nm to 760 nm sothat 712 nm light is detected by the infrared wavelength detector 820.The beam splitter, for example, includes dichroic mirror 815 reflectinglight having wavelengths exceeding 700 nm. For example, the dichroicmirror is stainless steel, though other suitable materials areacceptable. The infrared wavelength detector optionally includes anavalanche photodiode. Other suitable detectors, such as photomultipliertubes, are acceptable.

Optionally, light passing through the dichroic mirror 815 is subject toa bandpass filter 823, which passes, for example, light havingwavelengths in a range from 648 to 658 nm so that 658 nm light isdetected by the red wavelength detector 822. The infrared wavelengthdetector 822 optionally includes an avalanche photodiode. Other suitabledetectors, such as photomultiplier tubes, are acceptable.

Alternatively, light passing through the dichroic mirror 815 partiallyreflects off of a beam splitter through a standard bandpass filter 823,which passes 658 nm light to the red wavelength detector 822. The beamsplitter, for example, includes dichroic mirror 817, which reflectslight having wavelengths about 650 nm. Optionally, light passing throughdichroic mirror 817 is subject to a standard bandpass filter 827, whichpasses light having wavelengths about 645 nm to an optional side scatterdetector 825. Advantageously, the side scatter detector is optionallyused as a doublet discriminator so as not to mis-identify two or morebeads as a single bead.

I have also recognized that light path differences exist between themodified cuvette 800 and the various detectors described above in thealternative embodiment. Optionally, to compensate for the light pathdifferences, magnification lens 802 is focused to maximize a signalreceived at the infrared wavelength detector 820. Optionally, additionalstandard lenses are positioned between the dichroic mirror 817 and thered wavelength filter 823 and between the dichroic mirror 817 and theside scatter filter 827 to correct for light path differences thereto.

Optionally, light sources for the either the first described embodimentor the above-described alternative embodiment include a laser diode 830and/or a diode pumped laser 832, as shown, by way of example, in FIG.47. For instance, the laser diode 830 includes a laser diode emittingred light, such as 635 nm light, and the diode pumped laser 832 includesdiode pumped laser emitting a green light, such as 532 nm light.Optionally, one, two, or more lenses shaped the round beam from thediode pumped laser 832 into an elliptical spot on the cuvette. Theresulting elliptical spot, for example, includes a 20 micron by 60micron cross-section, wherein the major axis is horizontal.Advantageously, light from the laser diode on the cuvette is optionallyalready in the desired elliptical shape and need not require shapingoptics.

I have also recognized that performance of flow analyzers, such asdescribed herein, is facilitated by stability of the components thereof.I have determined that an optional optical assembly base or platform842, as shown, by way of example, in FIG. 47, advantageously enhancesstability of the components of the flow analyzer 25. For instance, a topof the modified cuvette 800 is effectively immobilized by an optionalstability bracket 840 connected to the optical assembly base frame 842.A bottom of the modified cuvette is effectively immobilized by a cuvetteholder top 190, described above and optionally connected to the opticalassembly base frame 842, though not shown in FIG. 47 for clarity.

Further to my goal of enhancing stability, the instant flow analyzer 25includes an optional U-block frame or assembly 812 affixed to theoptical assembly base frame 842. Optionally, one or more of the dichroicmirrors 815, 817 is adjustably connected to the U-block assembly 812 bya respective push-pull assembly, such as a screw tap and spring assembly835, 837. Screw tap and spring assembly 835 is optionally used to aim,automatically or manually, dichroic mirror 815 to the infraredwavelength detector 820. For example, the screw tap in the assembly 835pushes optional holders on the dichroic mirror 815, and the spring inthe assembly 835 pulls the dichroic mirror 815. Likewise, screw tap andspring assembly 837 is optionally used to aim, automatically ormanually, dichroic mirror 817 to the infrared wavelength detector 820.The holders include, for example, a stainless steel material, or othersuitable material.

I have further recognized that it is desirable to avoid sample fluidfrom entering the sample pump, for example, around the seals thereof. Ihave determined that a standard sample loop 862, as shown, by way ofexample, in FIG. 45, advantageously solves this very problem.Specifically, the sample loop 862 is dimensioned so that sample fluidenters the sample loop, but not the sample pump 100. By way ofillustration, referring to FIG. 45, sample fluid is drawn by the samplepump 100 into the sample loop 862. Then, for example, an optionalstandard three way valve 864 closes a valve to the sample injectionneedle, opens a valve to cuvette 70, 800, and pumps the sample fluidfrom the sample loop 862 into the cuvette.

I have also recognized that air bubbles in the sheath fluid streamhinder flow in the cuvette by clinging to the sides thereof, and aredifficult to remove. An optional de-bubbler 850, as shown, by way ofexample, in FIGS. 45 and 49, is situated between a sheath fluid supplybottle 852 and a fluid filter 854 downstream therefrom. The de-bubbler850 includes, for example, a linear polyethylene or polypropylenebottle, such as a NALGENE™ bottle. The bottle includes a vent or cap 856of which at least the top includes a breathable, substantiallywaterproof material, such as a GORE-TEX™ material. The de-bubbler 850includes an inlet 858 toward a top of the bottle and an outlet 860toward a bottom of the bottle. The inlet 858 is connected via a tube tothe sheath fluid supply bottle 852.

In operation, sheath fluid enters the de-bubbler 850 through inlet 858at a constant pressure, for example, at 7 psi. The pressure forces thesheath fluid out of the outlet 860. Air enters through the inlet 858,for example, when the sheath fluid supply bottle 852 is empty orsubstantially empty. When air enters through the inlet 858, there is aninsufficient pressure to drive the water lower and out of the outlet 860because the air escapes via the vent 856. The net effect is that no moresheath fluid is driven out of the outlet 860. When pressure sensor 95senses little or no fluid pressure, optionally the sample pump 100 isstopped. Advantageously, as such, the instant de-bubbler facilitates“on-the-fly” sheath fluid supply bottle changes without wasting orlosing sample fluid, namely, by expending sample fluid without using it.Optionally, substantially simultaneously, the control and analysissoftware 50 notifies the user to replace the empty sheath fluid supplybottle with a full one. Alternatively, or in addition, the flow analyzer25 includes an optional, externally visible indicator, for example, alight emitting diode, indicates to the user to replace the sheath fluidsupply bottle.

Other acceptable flow analyzers include those manufactured by LuminexCorporation of Austin, Tex., as disclosed in U.S. patent applicationSer. No. 09/102,034 to Applicant, incorporated herein by reference. Itis understood that, some commercially available flow cytometers, forexample, the Becton Dickinson FACScan flow cytometer include an originalcontrol computer typically supplied by the flow cytometer manufacturer.However, such a flow cytometer optionally is used alternatively with thepresent invention using, for example, a GPIO 2-position switch box. Theswitch box advantageously communicates with the original controlcomputer and the instant computer, permitting the user to select betweenthe original control computer and the computer described hereinbelow.Such a switch box advantageously permits retrofitting of an existingflow cytometer for use in the instant invention with limited, if any,effect on performance. Thus, commercially available flow cytometers areeasily used in combination with the remaining elements of the presentinvention, providing additional usage and/or versatility of the existingflow cytometer.

Flow Cytometer DSP Control

The flow cytometer has an external interface which allows a hostcomputer to communicate with it and control it. This communication usesa standard serial or parallel protocol.

Flow Cytometer Monitoring

The flow cytometer updates its external interface with a block ofparameters, which describes the current state of the cytometer. Thepertinent information includes, for example, the state of all of itsphotomultiplier tubes (PMTs), fluid levels, etc. The software optionallyreports the current status of these parameters to the user.

For example, the software optionally warns a user if the sheath fluidcontainer is empty and requires refilling. The software optionally alsowarns the user if the waste fluid container is full and requiresemptying. The software optionally notifies the user if apressure-related problem exists. A “Bad Link” message optionallyappears, if it is determined that the flow cytometer and the control andanalysis software or DSP Interface Card firmware is not communicatingproperly. The software optionally monitors the flow cytometer to ensurethat the setting match the calibrated settings required and set by theapplication.

Flow Cytometer Control

There are a plurality of flow cytometer parameters available to theserial or parallel connection interface card 15. The host computer, ifneeded, optionally changes at least one, most, or all of theseparameters. The software is optionally capable of changing any of theappropriate flow cytometer settings available through the externalinterface. The changed setting is relayed to the flow cytometer and isoptionally monitored as previously required. The software generallyissues its request for a parameter change to the flow cytometer promptlyafter entering an initial request.

Flow Cytometer Event Acquisition

The flow cytometer reports, some, and preferably all light scatterevents in the sample that pass through the flow cytometer and,optionally, are detected above a set threshold value. The softwarecollects these events for analysis.

The flow cytometer sends events across the serial or parallel connectioninterface, for example, as list mode data. By way of illustration,events are optionally sent in blocks of 15. Fewer than 15 events andmore than 15 events per block are optionally included in acceptable,alternative embodiments. Each event will contain the detected amount oflight at each photomultiplier (PMT) and an optional check sum to ensureproper transmission. Each PMT event is received as, for example, a tenset linear value or other appropriate set. The software optionallydiscards events that produce checksum errors.

Each event optionally contains the level of scatter for any or all ofthe following PMTs: forward scatter, side scatter, FL1, FL2, FL3, FLA,and FLW. FL1, for example, designates fluorescence channel one and isoptionally designed to capture light of a given color, for example,green. That is, the light first passes through a wavelength filter andis then collected by the FL1 PMT. FL2, for example, designatesfluorescence channel two and is optionally designed to capture light ofa given color, for example, orange. That is, the light first passesthrough a wavelength filter and is then collected by the FL2 PMT. FL3,for example, designates fluorescence channel three and is optionallydesigned to capture light of a given color, for example, red. That is,the light first passes through a wavelength filter and is then collectedby the FL3 PMT. The PMT values are optionally reported in, for example,linear and/or logarithmic form. By way of illustration the linear valuesof the PMTs is, for example, between 0 and 1023, and the logarithmicvalues of the PMT setting may be converted from its linear form into afour-decade log scale. In this illustration, there are 1024 fluorescencechannels. The software may be capable of processing, for example, 1000events per second.

Electronic and Filter Components of the Flow Analyzer

A standard power supply switch, for example, turns the instant flowanalyzer on and off. Optionally, the switch optionally automaticallydetects whether 110V or 220V voltage is being used. Otherwise, astandard adapter or external switch is optionally connected to theswitch, when operated in locations outside of the United States.

Additional equipment optionally includes a sheath fluid/air filteraccess door to removably cover a replaceable standard air filter 85 thatfilters air used to pressurize the sheath fluid. An optional standardfan is used to cool the flow analyzer. Optionally, the fan is locatedadjacent to a wall of the flow analyzer 25 to facilitate replacementthereof. Also, an optional air intake filter is located on the bottom ofthe flow analyzer. The air intake filter optionally is also configuredto facilitate regular or irregular cleaning thereof.

Fluidic Components of the Flow Analyzer

Disposal of, and prevention of exposure to, hazardous waste is alsofacilitated by the fluid dispensing and collecting components of theinstant invention. Advantageously, these components together comprise asubstantially completely integrated, closed fluidic system. Optionally,the instant invention includes a wall between fluidic components andelectronic components.

It is understood that human sample fluids may contain hazardousinfectious agents. Users of the instant invention should followappropriate biosafety procedures when handling the samples and theircontainers. The waste-fluid collecting components, advantageously,facilitate compliance with local, state, and federal biohazard handlingregulations as to disposing of biohazardous waste material.

The instant flow analyzer 25 optionally cooperates with standardlaboratory automation equipment. For example, as shown in FIG. 19, astandard integral pneumatic actuator and sample aspirator 205 moveablealong a vertical Z-axis optionally samples from wells of a standardmicrotiter plate 210 by incorporating a standard movable horizontal X-Yplate transport platform 215 underneath the flow analyzer 25. Forexample, an optional standard optical switch 845, as shown, by way ofexample, in FIG. 50, includes a tab which closes the switch when the armof the pneumatic aspirator is fully or substantially fully extended inthe down position. Optionally, when the optical switch determines thatthe arm is in the fully extended position, the sample pump 100 isautomatically shut off.

Optionally, travel of the pneumatic actuator is optionally interruptedby substantially any obstruction, thereby preventing damage or injury tothe user and/or the system. A standard communications port operativelyconnects the flow analyzer to the plate transport. Such a cooperatingaspirator advantageously provides true “walk-away” capability. Byextension, multiple flow analyzers, for example, eight or more flowcytometers, all connected to a single personal computer, are optionallyconfigured to work in parallel or in series to address the needs ofextremely high throughput operations.

Alternatively, the movable horizontal X-Y plate transport platform 215optionally supports the microtiter plate 210, instead of the flowanalyzer 25. In yet another embodiment, a plate transport optionally isobviated by an aspirator moveable in three directions, for example, eachmutually perpendicular from another. For example, the above-mentionedvertically moveable pneumatic sample aspirator 205 optionally isoperatively connected to at least two rack and pinion gear sets moveablein a horizontal X-Y plane. As another example, the above-mentionedvertically moveable pneumatic sample aspirator 205 is optionallyoperatively connected to a pneumatic piston moveable in a horizontalX-direction and to a pneumatic piston moveable in a horizontalY-direction.

For example, a sample aspirator carriage is optionally located on a faceof the flow analyzer or in communication therewith. The carriagetransports the sample fluid from the test tube to the cuvette of theflow cytometer. Upon operation, the carriage advantageouslyautomatically drops to the microtiter plate in place for sampleretrieval.

A standard sample fluid tube holder grips each microtiter tube in placebeneath the sample aspirator carriage. When using the plate transport,however, the sample fluid tube holder is removed for the sampleaspirator carriage to automatically retrieve sample from the platetransport.

A standard cover of the sample aspirator carriage optionally encases orcovers a standard fitting, such as a Cheminert fitting manufactured byValco Instruments Co. Inc. of Houston, Tex., which is optionallyloosened to allow adjustment of the length of a sample injection tube.

A standard sample injection needle or syringe is optionally housed in,for example, a stainless steel tube or other material. Optionally, thelocation of the sample injection needle is determined to facilitatereplacement thereof, if damaged. For example, the needle is optionallyaccessed via a front mounted door to facilitate replacement of the sealon the needle by a user. The sample injection tube or hose connected tothe sample injection needle carries sample fluid from the microtitertube into the interior of the flow analyzer 25. Advantageously, if theend of the tube becomes worn or frayed, the user may conveniently clipoff the frayed end and re-adjust the tube length.

A sheath fluid reservoir and a waste water reservoir optionally storessheath fluid 160 and/or waste water, respectively. The reservoirsinclude, for example, polyethylene bags 75, 110. Alternatively, thereservoirs include, for example, internally or externally situatedbottles. Advantageously, implementation of the reservoirs using externalbottles facilitates filling the same and permits a variety of sizedbottles to be used. A sheath fluid bag 75 is optionally pressurized,thereby forcing fluid into the system. As shown in FIG. 12, optionally,each bag includes a semi-permeable membrane patch level sensor, forexample, at a bottom thereof. The semi-permeable membrane patchincludes, for example, a standard GORE-TEX™ material, as manufactured byW. L. Gore & Associates, Inc. of Newark, Del. The sensor advantageouslyreleases air pressure, when fluid level drops below the patch, therebyadvantageously preventing any significant quantity of air from enteringthe system. When the electronic pressure sensor detects a drop inpressure, optionally, the operator is optionally prompted to replace thebag. A compartment containing the sheath fluid/waste water bag of theflow analyzer is optionally accessed by a user via an access door on theface of the flow analyzer.

Standard high volume connectors are optionally located on a side of theflow analyzer 25 to offer high volume users the option to connectdirectly to sheath fluid supply containers and waste water containers,for example, 20 L standard containers, rather than use the smallerinternally stored sheath fluid/waste water bag.

A standard pressure regulator is optionally located behind an accessdoor, for example, closest to the sample aspirator carriage. Thepressure regulator is optionally pre-set by a manufacturer thereof, andadjustments thereto optionally being made with the aid of TechnicalAssistance staff.

A syringe pump 100 optionally creates a vacuum that transports thesample fluid from the sample aspirator carriage and the flow cuvette.The syringe pump 100 is optionally located in the same compartment asthe pressure regulator. Because positive pressure need not be used toload the sample fluid into the flow analyzer 25, advantageously the useof dangerous aerosols are optionally reduced or avoided.

To achieve system miniaturization, smaller than ordinary fluidreservoirs are optionally used. However, doing so necessarily entailsconserving sheath fluid 160. A microcontroller optionally accomplishesthis purpose, for example, by calculating substantially the exactinterval prior to sample injection that flow must begin to achievehydraulic stability at any measured pressure. As soon as an assay iscomplete, flow is optionally halted, and any remaining sample fluid isoptionally diverted to the waste fluid bag.

Laser and Optical Components of the Flow Analyzer

As mentioned above, the flow analyzer 25 includes a co-planarlaser/detector array. Fluorescent signals are delivered to opticalamplifiers, such as avalanche photodiodes, where waveforms arephotoelectronically converted and amplified for analysis by the DSP.Standard algorithms derived from the wireless communications industryoptionally function with the DSP to greatly increase the signal-to-noiseratio and, therefore, sensitivity. The instant flow analyzer optionallyincludes interrupt driven, fixed rate sample acquisition into circularbuffers, which provide zero inter-event dead time. That is, suchcircular buffers 30 allow the processing of patient samples with wideanalyte concentration ranges without fear of losing rare events becauseof a slow processor.

Substantial miniaturization of the flow analyzer is optionally achievedusing standard diode lasers as the light sources 120 for the flowanalyzer 25. The laser/detector array, for example, two lasers and fourdetectors, are arranged in a co-planar configuration so as to allowclose working distances, and a single-filter light path to, for example,the avalanche photodiodes. Optionally, more than one filter operativelyconnects a detector and a corresponding optical amplifier, such as anavalanche photodiode.

The laser assemblies are optionally inaccessible to the user.Optionally, all required maintenance are performed by a system factory.

As mentioned above, advantageously, lenses, mirrors, and detectorscommon to current flow cytometers for light collection are optionallyreplaced with a hexagonal cuvette, in the instant invention. Such ahexagonal cuvette provides a flat air-to-glass interface for the laserdiodes and the detectors.

Optional Standard Lab Equipment

Equipment, for calibration of the instant invention, include, forexample, a standard bath sonicator, probe sonicator and/or a standardvortex. Additional materials for calibration optionally include standardcalibration beads, such as FlowMetrix Calibration Microspheres 41-55001,and a standard read tube, such as a FACS-compatible read tube.

System Operations

The system is powered up to a known state and, optionally, indicateserror conditions if errors occur during the power-up. A user powers up aflow cytometer, and optionally its host computer, including anyperipherals. At the end of the power up sequence, the software is in astate such that the user can begin to operate the system and softwarewith proper controls available. The software advantageously andoptionally indicates whether any flow cytometer errors occurred duringpower up and/or whether the flow cytometer communications were notavailable.

The software is optionally configured to allow a user or operator to setup and define a new sample diagnostic run. The user optionally invokes anew run via a graphical interface of the application. The software, forexample, presents the user with an entry form containing fieldspertinent to a new run. By way of illustration, the form includes afield for the user's name, a field for the number of samples containedin the run, and/or a field for the description of the run.

Standard maintenance and calibration procedures for the flow cytometerare optionally followed. By way of example, a calibration procedure forthe instant invention optionally includes adjusting PMT voltages suchthat microspheres will advantageously produce similar readings acrossdifferent flow cytometers of the same or different brands and/or models.For example, the user processes a sample containing a standardcalibration solution having, one, two, three or more calibration beadtypes. Known, predicted, peak measurements, for example, side scatter,of FL1, FL2, and/or FL3 are targets for the calibration process, foreach calibration bead type. Advantageously, the software automatically,or by user demand, makes adjustments to the flow cytometer, while thecalibration sample is running until these target goals are achieved orwithin a feasible time period from the conclusion of the calibrationrun. That is, for example, the software is optionally calibrated usingautomatic adjustments implemented in the software such that all beadtypes are recorded in their predicted regions. To this end, the softwareoptionally adjusts the PMT so that the measured peaks of the calibrationbad types come to within one, two, three, four, five, or more channelsof their target values. When using avalanche photodiodes as the opticaldetectors, optionally, calibration software advantageously computes orapplies a temperature compensation table so as to ensure that theavalanche photodiodes have substantially constant gain at all orsubstantially all temperatures in a standard operating range.

Method of Operation Overview

An illustrative general method of operation of the diagnostic systemincludes the steps as shown, by way of example, in FIG. 41. In stepS100, a biological sample is run through a flow analyzer until usertermination, sample shortage, or sheath fluid shortage. In Step S110,the presence and quantity of one or more analytes of interest in thebiological sample substantially simultaneously to the running step.Acceptable alternative embodiments of the method of operation areoptionally found in U.S. Ser. No. 09/102,034 to Applicant, and U.S. Ser.No. 09/000,286, to Applicant, Jerrold R. Fulton, and Mark B. Chandler,both references being incorporated herein by reference.

Control and Analysis Software Overview

The control and analysis software controls operation of the flowcytometer and performs real-time digital analysis of one or morebiological samples for one or more analytes of interest, simultaneouslyor substantially simultaneously including sequentially. Real-timeanalysis according to the instant invention is intended to include, butis not limited to, determining an identity and quantity of, at least oneof and, optionally, each analyte of interest in a biological sample bysubstantially simultaneously or substantially contemporaneouslyperforming the following steps or sub-steps. Microsphere or particleclassification data and reactant-analyte complex measurement data arecollected. For example, each microsphere are classified according to itssubset of microspheres. The amount of reactant-analyte complexassociated with each subset of microspheres are quantified.

The software operate on any standard operating system platform, forexample, Microsoft Windows 95 operating system located on a personalcomputer, standard network, or other global network. Alternate operatingplatforms include Solaris, Linux, Java, Mac OS, and/or IBM OS/2, forexample. A controller optionally integrates the software and thecommunications interface to the flow analyzer 25.

The control and analysis software 50 according to the present inventionincludes two modules, as illustrated in FIG. 20. One module is calledData Acquisition 300, and the other one is called Multiplexed Analysis305.

The control and analysis software 50 communicates with appropriatestandard libraries. These libraries, for example, include an applicationprogramming interface library, such as a LumAPI library, and/or amathematics library, such as a MHMath library, both of which beingdescribed hereinbelow.

The control and analysis software 50, for example, includes a standardinterface used to collect data from the flow cytometer 25 via the serialor parallel connection interface card 15. The control and analysissoftware 50 initializes and obtains status information from the flowcytometer 25. It also permits user entry of assay kit information,including bead subset data. For example, if an Immunoglobulin G, A, MIsotyping Assay were intended, standard kit information concerning theIg GAM Assay Kit produced by Luminex Corporation is optionally entered.

The control and analysis software 50 instructs the flow cytometer 25 toprocess a biological sample. The software 50 displays a graph of anappropriate linear trend line for prediction. A Logit Logtransformation, for example, is used to calculate such a linear trendline for prediction. Such a calculation is as follows

${{Logit}\left( \frac{MIF}{MIFo} \right)} = {\ln\left( {\left( \frac{MIF}{MIFo} \right)/\left( \frac{1 - {MIF}}{MIFo} \right)} \right)}$where MIF_(o)=negative control, and MIF=control The predicted valueusing a Logit Log transformation, is

$10^{\ln{({{(\frac{MIF}{MIFo})}/{(\frac{1 - {MIF}}{MIFo})}})}} - \frac{y\;{intercept}}{slope}$

The intercept and/or slope values are calculated using the polynomialtrendline routines in a standard mathematics library, such as MHMath.The software 50 produces a report indicating success or failure ofdetection of analytes of interest in each sample and, optionally,reasons for failure. The report is, for example, in tabular form. Thereport, for example, includes a header with pertinent run informationsuch as data, operator, and/or a description of the run. For example,the report optionally includes concentration levels of large G, A, M,for patient samples, if an Ig G, A, M assay were run. The software 50,for example, produces an x-y graph displaying

${Logit}\left( \frac{MIF}{{MIF}_{o}} \right)$on the y-axis and

${Log}\left( \frac{mg}{d\; L} \right)$on x-axis. A standard trend line from, for example, the first controlvalue of a concentration level to, for example, the fifth control valueof a concentration level is optionally drawn.

The control and analysis software 50 guides an operator through stepsnecessary to complete a diagnostic run. The software, for example,allows the operator to define a new sample diagnostic run. The userinvokes a new run through the software interface. The system dictatesthe order in which the samples are processed, preventing the user fromrandomly selecting which sample is processed during a run. Optionally,the system permits the user to determine the order of the samples to beprocessed. The operator or user optionally manually loads the flowcytometer 25 or allows automatic sample acquisition via a movablemicrotiter plate transport platform 215.

Data Analysis

The software advantageously includes a method by which a user initiatescapture of flow cytometer events.

In use, there may be some spectral overlap in the excitation curves ofone or more bead identification fluorescent dyes and one or moreanalytes of interest identifying dyes. For example, a green fluorescentdye identifying an analyte of interest and an orange fluorescence dye,at least partly identifying a bead subset, may suffer from spectraloverlap of the respective excitation curves. Alternatively, for example,an orange fluorescent dye identifying an analyte of interest and redfluorescent dye and an infrared fluorescent dye identifying a beadsubset are used. In such a case, for example, the orange fluorescent dyeand the red fluorescent dye may suffer from spectral overlap of therespective excitation curves.

To correct this, a standard color compensation function to account forthe amount of green fluorescence present in the orange reading andvice-versa is optionally included in the software 50. Color compensationoptionally is applied immediately to the events as received using astandard color compensating algorithm. Thus, events optionally areadjusted to indicate their actual levels of orange and green inreal-time. The performance of the color compensation process optionallyis less than 1000, equal to 1000, or greater than 1000 events persecond.

Advantageously, the system optionally ignores events due to aggregatedbeads and other events that do not correspond to the size of a singlebead. For example, the system optionally ignores events by gating theside scatter collector to a narrow range defined by the assay after theevents have passed through the color compensation function. Optionally,the output of the gating process optionally includes eventscorresponding to a uniform shape of a single bead of known diameter, forexample, 5.5 μm. Optionally, the performance of the gating processoptionally conforms to a rate of less than 1000 events per second, 1000events per second, or more than 1000 events per second.

Bead Identification

The software 50 collates or categorizes bead types based, at least inpart, on color content. Naturally, in addition, the system optionallycategorizes bead types based on other or additional factors, such assize and magnetic coding.

Bead identification includes a function of the fluorescence channels FL2and FL3 parameters for a given event. Advantageously, I predefinedregions for each bead in the system described in an x-y grid. The FL2values, for example, make up the x-axis, the FL3 values, for example,make up the y-axis. The units along the axes optionally are units offluorescent channels or fluorescence. For example, each axis includes1024 fluorescent channels. Plainly, each axis alternatively can havemore than 1024 fluorescent channels as dyes with greater emissionspectrum definition become available or as greater emission spectrumdefinition becomes possible with present, standard dyes. The beadidentification process, for example, maps an event to a specific beadsubset identification number and optionally dismisses the event as notbeing a valid bead. A classified bead includes a bead identificationfrom, for example, the FL2 and FL3 values and a FL1 measurementdesignating the presence and/or amount of analyte of interest on thebead.

The event collection and bead identification process is optionallycapable of identifying less than 1000 beads per second, 1000 beads persecond, or greater than 1000 beads per second.

Calculating Bead Statistics for Each Bead

Once the software has identified the event as belonging to a specificbead classification, bead statistics are optionally calculated.

For example, a count statistic optionally tracks the number of beads ofa given bead subset classification that have been acquired during thecurrent sample. Plainly, the sum of all bead counts must equal thenumber of beads collected in the current sample. Again, performance isoptionally less than, equal to, or greater than 1000 events per second.

A FL1 Linear Mean determination optionally is performed after an eventis identified as belonging to a specific bead classification. FL1 LinearMean, for example, equals the sum of all FL1 linear values of a givenbead type divided by the count of beads collected for that type.Performance, for example, is as discussed above.

A FL1 Linear Standard Deviation is optionally calculated. It, forexample, includes the standard deviation calculation for the linear FL1values of a given bead type as is done for each bead type in the currentsample after an event has been identified as belonging to a specificbead classification. Performance, for example, is as discussed above.

A FL1 Linear Coefficient of Variation (Linear CV) optionally is alsocalculated. It, for example, includes the standard deviation of linearFL1 values as represented as a percentage of its linear mean. Beforeperforming this calculation, the software optionally calculates thelinear standard deviation and linear mean measurements for the givenbead classification. The linear CV measurement for each bead type in thecurrent sample is then calculated. Performance is optionally asdiscussed above.

A FL1 Linear Peak optionally is also calculated. It, for example,includes the linear FL1 value having the most occurrences during thecurrent sample for each bead type, after an event is identified asbelonging to a specific bead classification. Performance optionally isas discussed above. Linear peak measurement equals, for example,integers from 0 to 1023, assuming 1024 fluorescent channels. Plainly,acceptable linear peak measurement values are more or less depending onthe number of fluorescent channels.

The above-mentioned FL1 calculations are, for example, used to ascertainstatistically significant concentration levels of an analyte ofinterest. Optionally, such ascertaining includes comparisons tobackground FL1 concentration levels in a sample.

Data Acquisition Module

The Data Acquisition module 300 acquires fundamental data coming fromthe machine, and is not necessarily bead specific. That is, the DataAcquisition module 300 yields statistics for, by way of illustration,the side scatter channel and/or the different fluorescent channels, suchas, Fluorescent channel 1 (FL1), Fluorescent channel 2 (FL2), andFluorescent channel 3 (FL3) using standard techniques. The fluorescentchannels represent the state of fluorescence of a detected bead. Thus,the Data Acquisition module 300 optionally yields data that relate to anevent. For example, data provided by the Data Acquisition module 300includes, for example, a cell that has been stained, a fluorescentlylabeled bead, or an indication that no beads are present.

The Data Acquisition application module 300 includes, for example, asimplex analysis application module or option for use with, for example,single bead sets or other particles or cells. Such a simplex analysisoption facilitates the initial setup of an experiment when settings,gates, and/or reagents, for example, have not yet been determined.

The simplex option includes one or more templates, files, or foldersthat contain stored PMT settings, gates, reagents, bead set values,detection regions, and/or spectral overlap compensation settings for usein experiments. By way of illustration, it is to be understood that thephotomultiplier settings are optionally replaced with avalanchephotodiode settings or a combination of photomultiplier and avalanchephotodiode settings. The simplex analysis option optionally includes aprovision whereby a user stores all events, gated and non-gated, to afolder or only those events passing through designated gates. The useroptionally selects an established template, establish settings and savethem as a new template unique to the user's needs, and/or proceed to theexperiment without creating a template. As to the third selection, whenthe experiment is completed, the user optionally saves the used settingsas a new template or folder. Settings, for example, include assaydescription, assay operator, number of gated events to collect persample, flow rate, and/or number of samples. Upon completion ofexercising the simplex analysis option, the software is optionally readyto acquire and analyze data from the flow analyzer.

Features Common to Multiplexed Analysis Module and the Data AcquisitionModule

Fluorescent channel 1, Fluorescent channel 2 and Fluorescent channel 3,for example, include fluorescent signals of same or differentwavelengths. A light Source, such as a laser, excites the fluorescentsignal at one wavelength, but the fluorescent signals then emitfluorescence at, for example, different wavelengths detected atdifferent windows or filter units. The fluorescence data is inputtedinto the software for multiplexed analysis. The invention classifies adata event as, for example, a bead or cell data unit, using, forexample, gating on a forward light scatter channel and/or a side lightscatter channel. For example, the beads are identified by using a gatingfilter calibrated by a user or a manufacturer to appropriately identifya range of forward and/or side light scatter data associated with a beadtype. Using such a filter on any incoming event, for example, if a sidelight scatter channel does not read a meaningful event, then theinvention optionally throws out the event and does not collect data.

Bead data are placed or stored in a logical bucket, or a database.Signals from the bead are passed through, for example, the side lightscatter channel. If the bead (beads) passes the side scatter filter,then the bead type is optionally determined. The FL2 and FL3 channelsmay yield varying signals, optionally corresponding to predefinedregions associated with respective bead types, for example. So if, forexample, FL3 on the Y axis and FL2 on the X axis are plotted in aspectral table, each bead optionally has a predefined spectral region.This predefined region optionally includes an elliptical region of wherebeads of a given subset are designated to fall into, for example, asshown in FIG. 21. These regions may suffer from spectral overlap becauseof FL1 signals, for example, thereby rendering the bead regionsindistinguishable. By way of illustration, each bead region has analphanumeric identifier. Each identifier optionally corresponds to arespective analyte of interest. If a processed signal is identified asbelonging to a particular bead region, then automatically, itsalphanumeric identifier and analyte of interest are known, andappropriate tracking variables are optionally updated.

So, while these events are processed in real-time, color compensation tocorrect spectral overlap are optionally performed to determine the realfluorescent values for FL2 and FL3. Then, the bead fluorescence arematched with the spectral table to determine whether they fall into oneof the predefined bead subset regions. If the fluorescence does notmatch, then the data is not included in the statistics. Examples oferrors include beads that fall slightly out of the regions, trash,spurious noise, and/or the like.

It is to be understood that the above processes are performed using thesimplex option or a multiplexed analysis option. Data are recorded to,for example, Flow Cytometry Standard (FCS) files for list mode analysisand/or spreadsheet compatible files, such as CSV files, for spreadsheetanalysis.

By way of non-limiting illustration, the Data Acquisition applicationmodule 305 and/or the Multiplexed Analysis application module 305includes a graphical display 320, for example, as shown; by way ofexample, in FIG. 22 and as described hereinbelow. For example, thegraphical display 320 includes, as shown, by way of illustration, inFIG. 23, a title bar 325 indicating, for instance, the experiment folderor template name currently open. The display includes, as shown, by wayof illustration, in FIG. 23, a main menu bar 330 having a number ofoptions, such as pull-down menu options. For example, the optionsinclude one or more of the following.

Title and Main Menu Bar

The Main Menu Bar 330 optionally includes a “Folder” option 335, which,in turn, includes one or more of the following choices. A “New” folderchoice 340 provides means or functionality for creating a new experimentfolder using standard techniques that are provided, for example, in awindows-like environment. This means also include choosing a newexperiment folder as shown, by way of illustration, in FIG. 24. Thismeans also include choosing a folder or template, choosing a locationfor the new experiment, and/or naming the folder in which the data andexperiment settings will be stored. An “Open” folder choice providesmeans for opening an existing folder. A “Save” folder choice providesmeans for saving a folder with a current name. A “Save As” folder choiceincludes means for saving the folder with a new name. A “Print” folderchoice provides means for selecting which components to be printed by,for example, selecting or checking the appropriate box, such as, for aresults table, a dot plot, and/or a histogram. A “Create Template”folder choice includes means for saving PMT settings, gates, regions,etc., as a template file to be used in future experiments. Note thatexperimental data need not be stored through this option, but rathertemplate settings.

A “Main” option 345, as shown, by way of illustration, in FIG. 23,includes means for returning the user to an opening screen or window forthe control and analysis software.

A “Flow Analyzer” or “Cytometer” option 350, as shown, by way ofillustration, in FIG. 23, includes one or more of the following choices.A “Machine Set Up” choice 355, as shown, by way of example, in FIG. 25,provides means for selecting among a number of flow analyzers havingcharacteristics included in the software. A “Calibrate” choice includesmeans for calibrating the flow analyzer and/or stores in a data file theinformation obtained. By way of example, this operation is performedadvantageously immediately upon installation of the analyzer and, forinstance, monthly thereafter. A “Connect” choice includes means forestablishing an initial connection between the software and certain flowanalyzers, such as the Becton Dickinson FACSCalibur model. Note thatother flow analyzers, such as the Luminex Corporation's LUMINEX100™model, do not require this procedure.

A “Sample” option 360 provides means for displaying data from a previousexperiment. For example, the “Sample” option includes a “Load Data”choice. After an existing folder is opened and one or more samples arehighlighted, the “Load Data” choice, which includes means for retrievingdata histograms, and/or dot plots to a display or printer. Optionally,this “Load Data” choice includes means for displaying dataincrementally.

A “Help” option 365 includes means for getting help relative to commonprocedures, errors, and/or frequently asked questions.

Results Table

The graphics display for the Data Acquisition application module and/orthe Multiplexed Analysis application module includes a graphical resulttable 370, as shown, by way of illustration, in FIGS. 22 and 26. Theresults table 370 is optionally displayed upon first entering themodule. The Results table 370 includes means for displaying datacollected during an experiment as the cytometer acquires it.

The Results table 370 includes one or more of the following features. A“Start” option or virtual button 375 provides means for togglingacquisition of data via the flow analyzer 25. Optionally, the “Start”button, for example, includes a graphical indication of operation, suchas by downward movement of a bead through an examination zone. That is,for example, although a flow cytometer 25 is set to “Run”, optionally,no data is acquired until the “Start” button 375 is clicked or selected.Advantageously, such a feature permits a user to leave the flowcytometer 25 on “Run” between biological samples and to resume dataacquisition upon selected the “Start” button 375. Optionally, with thesample table on the probe and the flow cytometer 25 on “Run”, the samplecontinues to pass through the flow cytometer 25. In such a manner, dataoptionally is not collected, although the flow cytometer 25 continues touse sheath fluid and/or produce waste.

The Results table 370 optionally includes a row or column indicating aname or number of one or more samples and/or a background or baseline.The Results table 370 also optionally includes one or more columns orrows each labeled for a respective bead region, or analyte of interest.Each such column or row would track the number of events processed andidentified as one of the respective bead regions. The Results table 370also optionally includes an “Events” column or row, tracking the totalnumber of events processed for a sample or a background test.

A “Description” user entry option or virtual button 380 includes meansfor recording and/or displaying a description of the experiment. The“Description” option 380 optionally includes, for example, input takenfrom the template selected.

An “Operator” user-entry option or virtual button 385 includes means forrecording and/or displaying an individual or team conducting theexperiment. The “Operator” option 385 includes, for example, relevantinformation taken from the selected template.

A “Clear All” user-entry option or virtual button 390 includes means forclearing a display, such as a screen, of, for example, all data andgraphs, and optionally delete associated files. Optionally, the “ClearAll” option 390 further includes means for prompting a user with awarning message prior to execution such as, “Continuing will clear thetable, graphs, and collected data files. Do you want to continue?”

A “Statistic” user-entry option or virtual button 395 includes means foroffering one or more of the following statistics to be displayed foreach parameter in the table: mean, coefficient of variation (StandardDeviation/Mean×100), Count for Gated events, for example, if gates areset), Peak value, Standard Deviation from the Mean. The offering meansinclude, for example, a pull-down menu.

A “Show as channel data” user-entry option 400, such as a check box, orvirtual button, includes means for allowing a user to select which typeof data will be displayed in the table. This option, for example, doesnot affect storage of data, but rather affects the display of data.Optional types of data, for example, include channel data, such as, datadisplayed in channel numbers regardless of whether the signals werecollected from the flow analyzer in, for example, linear or log mode.Alternatively, optional types of data, for example, include data values,such as, data collected in log value if signals were collected using logamplification or in linear value if signals were collected using linearamplification.

An “Events to collect” user-entry option or virtual button 410 includesmeans for indicating the number of events to be collected for eachsample as set in, for example, an Assay information display or window.For example, this option optionally includes a column or row indicatinga corresponding parameter such as, Forward Scatter (FSC), Side Scatter(SSC), Fluorescence channel 1 (FL1), Fluorescence channel 2 (FL2),and/or Fluorescence channel 3 (FL3). The option 410, in addition oralternatively, optionally includes a total number of gated eventscaptured for a given sample.

The Results table 370 optionally includes an “Edit” user-entry option orvirtual button 415 to open an Assay Information graphical display window420, as shown, by way of example, in FIGS. 27 and 28, to edit settingstherein. The Assay Information window includes, for example a Generaltab or frame 421. For example, the General tab 421 includes an Assay onDescription entry, an Operator entry, a Number of Events entry, a Numberof Samples entry, and/or a Flow Rate entry. The Output tab 423, forexample, includes a check box for automatically exporting data to aspreadsheet upon closing the software and/or a check box for recordingall gated and non-gated events.

System Monitor

The graphical display for the Data Acquisition application module and/orthe Multiplexed Analysis application module optionally includes agraphical System Monitor 425, as shown, by way of example, in FIG. 29.For example, the System Monitor 425 includes a vertical information barlocated on the left or right side of the display or screen. Plainly, theSystem Monitor 425 alternatively includes a horizontal information barlocated on the top or the bottom of the display or screen. Optionally,the System Monitor 425 is non-contiguous, whereby portions thereof arelocated in areas of the display or screen convenient to the user.

The System Monitor 425 include one or more of the following features. A“System Status” display 430 includes means for displaying current statusof the operation and/or warnings, such as “Ready”, “Standby”,“Pressure”, and/or “Bad Link”. Optionally, the System Monitor 425includes an “Events” display 435 for displaying a total count of events,a count of gated events, a count within a set region, and/or a number ofmissed events or events for which data could not be collected.Preferably, the number of missed events should always equal zero.Optionally, the System Monitor 425 optionally includes an “Events/Unittime” display, such as “Events/second” display 440 for displaying eventsrecorded per second, thereby indicating the concentration of beads. The“Events/second” display 440 includes a total concentrating value, aconcentration value for gated events, and/or a concentration value for aset region. Optionally, the System Monitor 425 includes an“Events/liquid unit” display, such as an “Events/μL” display 445, fordisplaying events per microliter based, for example, upon the flow rateset by selecting the edit option on the Results table 370.

Histogram Frame

The graphical display for the Data Acquisition application module 300and/or the Multiplexed Analysis application module 305 includes auser-selectable frame having one or more selectable feature tabs. Onesuch feature tab includes one or more data graphs such as a histogramtab or frame 450, as shown, by way of illustration, in FIG. 30. The datagraph optionally provides a graphical display of the real-time datagathered or a graphical display of data gathered and shown on atime-delayed basis. The data graph cooperates with other elements of thegraphical display, such as, the “show as Channel Data” option or checkbox 400 in the Results Table 370.

By way of illustration, to view the assay results in linear channels auser selects or checks the “show as Channel Data” check box 400 in theResults Table 370. Such optional default data are reported in the modein which it is collected and stored if the “show as Channel Data” checkbox is not checked. Optionally, at least one of the features areselected by a virtual pointer, such as by placing a mouse pointer on thehistogram tab 450 and clicking on the right or left mouse button for adesired feature. The histogram tab 450, which for example, is a defaultdata graph type, includes one or more of the following features orfunctions. It is, of course, understood that the below-mentioned X and Yaxes are interchangeable as may be beneficial to the user.

An X-Axis function provides means for selecting, by a user, whichparameter, will be displayed on the X-axis of the histogram. Optionalparameters include, for example, forward scatter, side scatter,Fluorescence channel 1, Fluorescence channel 2, Fluorescence channel 3,Fluorescence channel 4, Fluorescence amplitude, and/or Fluorescencewidth.

A Gate function includes means for adjusting, by a user, the gate in thehistogram or other data graph. Once a gate is set, data reflected in theResults Table 370 are processed through that gate and/or through the DotPlot, as mentioned above, if set.

A Switch function includes means for switching the histogram to displaythe parameter in which the acquisition gate has been defined.

A Create function includes means for creating a new gate. The meansinclude adjusting a new gate by positioning a mouse pointer, forexample, on a gate border, such as a dotted vertical line, selecting thedotted line, and dragging it to a new desired position to form a borderof the new gate.

A Delete function includes means for deleting or removing a currentgate.

An AutoScale function includes means for setting, by a user, one or morehistograms, such as for fluorescence Channel 1, Fluorescence Channel 2,and/or Fluorescence Channel 3, to the same Y-Axis scale.

A Set Scale function includes means for setting, optionally manually, bythe user, the Y-Axis scale. The user sets and enters a maximum number ofevents, using this means.

The frame includes a user-selectable optical amplifiers or PMT's frameor tab 455, which includes means for controlling the photomultiplier oroptical amplifier settings of the flow analyzer as shown, by way ofexample, in FIG. 31. Optionally, changes made via this means areoptionally reflected on a control panel of the flow analyzer.

The PMT tab 455 includes a Channels option 460. This option includesmeans for selecting a parameter, such as forward scatter, side scatter,Fluorescence Channel 1, Fluorescence Channel 2, Fluorescence Channel 3,and/or Fluorescence Channel 4. Fluorescence Channel 4 is used, forexample, with an appropriately modified FACS Caliber modelBecton-Dickinson flow cytometer. Once the parameter is chosen, detailsthereof are optionally displayed in the frame. Optionally, all changesto the parameters are stored irrespective of whether they are displayed.

The PMT tab 455 optionally includes a Data Mode option 465 includingmeans for selecting, by a user, either linear or log mode. The PMT tab455 optionally also includes a Stage option 470 having means forselecting, by a user, a Detector feature for adjusting the voltage of aoptical amplifier, such as a PMT, and/or Amplifier feature for adjustingthe linear gain if the Linear Data Mode is selected. The PMT tab 455optionally includes a Level option 475 having means for establishing theoptical amplifier or PMT voltage or gain for the selected channel,depending, for example, on the Data Mode 465 and/or Stage 470 selected,for example, using a graphical slide.

The PMT-tab 455 optionally also includes a Threshold option 480 having achannel selector and/or a threshold slider. The channel selectorincludes, for example, a pull-down menu allowing a user to select anappropriate parameter to be adjusted using the threshold slider. Forexample, a default parameter is side scatter. The threshold sliderincludes means for adjusting the channel number for the threshold if theselected parameter. That is, by manipulating the slider, the userestablishes a minimum channel limit for detection of an event.

The frame optionally also includes user-selectable Compensation tab 485,as shown, by way of example, in FIG. 32. The Compensation tab 485includes means for setting, by a user, percentages (for example, from 0%to 99%) of software spectral overlap compensation override for one ormore of the fluorescence channels offered using the threshold slider.The Compensation tab 485 also optionally includes a selection or checkbox 490, for example, for selecting the software compensation override.The feature also includes the fluorescence channels available forsoftware compensation override. To this end, by way of illustration, ifthe software compensation override check box 490 is not checked orselected, the compensation levels established by the flow analyzerhardware remains in effect.

Each fluorescence channel includes a selectable option button, forexample. When an option button for a channel is selected, thecompensation level is optionally displayed, for example, on the tab.Optionally, all of the established compensation levels are sorted evenwhen not displayed on the tab. The Compensation tab 485, for example,includes one or more of the following fluorescence channel ranges, whichare adjustable by, for example, a graphical compensation slide 495compensation:

-   % FL1%-FL2 (which decreases interference of fluorescence from    Fluorescence channel 2 into Fluorescence channel 1)-   % FL2%-FL1 (which decreases interference of fluorescence from    Fluorescence channel 1 into Fluorescence channel 2)-   % FL2%-FL3 (which decreases interference of fluorescence from    Fluorescence channel 3 into fluorescence channel 2)-   % FL3%-FL2 (which decreases interference of fluorescence from    Fluorescence channel 2 into Fluorescence channel 3)-   % FL2%-FL1 (which decreases interference of fluorescence from    Fluorescence channel 1 into Fluorescence channel 2)-   % FL1%-FL3 (which decreases interference of fluorescence from    Fluorescence channel 3 into Fluorescence channel 1)

graphical color compensation slide 495, for example, permits a user toset percentages for the above channel ranges, such as from 0% to 99.9%.The frame optionally includes a Doublet Discrimination Module (DDM) tab500, as shown, by way of example, in FIG. 33, having means fordistinguishing between singlets (single beads) and doublets (two or morebeads non-purposely affixed to each other). The DDM tab 500 includes,for example, a selectable checkbox 510 for enabling the feature.Optionally, the DDM checkbox 510 is optionally always checked, or ischecked as a default feature, if the associated flow analyzer 25 iscapable of detecting doublets. The DDM tab 500 optionally includes anApplied Collector feature 515 having means for selecting, by a user, aappropriate parameter for pulse processing. By way of illustration, auser selects one or more channels, such as Fluorescence channel 1,Fluorescence channel 2, and/or Fluorescence channel 3, to obtain morespecific data. The DDM tab 500 optionally includes an amplifier feature525 having means for setting, by the user, a gain via a virtual slider,for example. By way of illustration, an FL-A slider optionally enables auser to set the signal area for the channel selected in the AppliedCollector feature. Similarly, by way of example, an FL-W slider 530optionally enables a user to set the signal width for the channelselected in the Applied Collector feature 515.

Dot Plot Frame

The graphical display for the Data Acquisition application module 300and/or the Multiplexed Analysis application module 305 optionallyincludes another frame for displaying a real-time, two-parametergraphical display such as a Dot Plot graphic display 535 of thecollected data, as shown, by way of illustration, in FIG. 34. It isunderstood that this display is optionally time-delayed and/or includesmore than two parameters. The Dot Plot graphic display, for example,depicts the data as accumulations of tiny dots, each dot representing adata point based on the two parameters, for example.

The Dot Plot graphic display 535 includes one, two, or more choices forthe display of the data. For example, an optional choice includes aDensity Dot Plot 540 having means for displaying constant accumulationof events with increasing or decreasing density depicted by, forexample, contrasting or differing colors, shading, and/or hatching. TheDot Plot graphic display. 535, alternatively or in addition, includes aDecaying Dot Plot 545 having means for displaying a number, for example,1, 10, or 100 or more, of the most recent events acquired by the flowcytometer. Optionally, the Decaying Dot Plot 545 is updatedcontinuously, i.e., in real-time, as data are collected, or updated on atime-delayed basis.

Additional optional features of the Dot Plot Frame 535 include one ormore of the following. They, for example, are selected by a virtualpointer, such as via clicking on the right or left mouse button in theDot Plot frame. For instance, optional X-Axis and/or Y-Axis choicesinclude, for example, respective pull-down menus to set one or more ofthe following parameters: forward scatter, side scatter, Fluorescencechannel 1, Fluorescence channel 2, Fluorescence channel 3, FluorescenceArea, and/or Fluorescence Width. Alternatively, or in addition, the usermay define or re-define these parameters by selecting, for example, byleft or right clicking on a mouse, on the X-Axis or the Y-Axis.

A Region choice is an optional Dot Plot Frame feature. Optionally, it isavailable only in the Data Acquisition application module. The Regionchoice includes means for establishing, by a user, regions for viewingdata specific to the user's needs. In turn, the Region choice includesone or more of the following options. A Show option includes means forshifting, when selected, the display to those parameters in which thespecified region exists. A Create option includes means for creating, bya user, a new region by, for example, one or more of the followingsteps. A mouse pointer, for example, is moved to an area in the Dot Plotwhere the new region is to be created. A left or right mouse button isdepressed while dragging the associated cursor over the appropriate areaencompassing the desired region. Optionally, the encompassed regionoptionally changes color, for example, from gray to white, indicatingthe location and area of the new region. To modify an establishedregion, for example, a keyboard key, such as, the shift key, and theleft or right mouse button is optionally held down while the mouse isdragged to alter the region.

A Density Plot Options feature 550, as shown, by way of example, in FIG.35. The options feature 550 includes means for adjusting the scaleand/or other features of the Density Dot Plot, and/or means foreliminating data values determined to be insignificant to the display.For example, the Options choice includes an entry selection or checkbox555 for Filter Levels, which includes means for filtering out eventsthat fall below a desired level. For instance, if the Filter Levelscheckbox 555 is not checked, a “Display above level” option 560, forexample, is optionally not available, and a Level Multiplier option 565,for example, has no effect. The “Display above level” option 560, inoperation, includes means for setting, by the user, the level at whichevents will be displayed in the dot plot 535. Events below this levelare optionally ignored. This level is a number, which, for example,include an exponent of the Level Multiplier 565, and is optionally set,for example, between 1 and 8, although levels greater than 8 are alsopossible. The Level Multiplier 565 includes means for establishing, bythe user, the necessary base number of events that must fall within aregion before being displayed.

By way of illustration, if the Filter Level checkbox is checked, the“Display above level” option 560 is set at 3, and the Level Multiplier565 is set at 2, events optionally are not displayed until 8 or moreevents have been registered. According to this illustration, a firstcolor level in the dot plot display 535 is at 8 events, a second colorlevel is at 16 events, a third color level is at 0.32 events, etc.

Optional Additional Features of the Multiplexed Analysis Module

The Multiplexed Analysis Module 305 optionally adds to the functionalityavailable in the Data Acquisition module. Optional features of theMultiplexed Analysis Module 305 are described as follows.

The Multiplexed Analysis module 305 includes, for example, an AssayInformation graphical display window 420 having one or more featuretabs, as shown, by way of illustration, in FIG. 36. For example, inaddition to or alternative to the General tab and the Output tabdescribed above, an optional Bead Set tab 570 includes one or more ofthe following options.

The Bead Set tab 570 optionally includes an “Available” option 575having means for listing one or more, up to all, available bead sets.The Bead Set tab 570 optionally includes a “Selected” option 580 havingmeans for storing for ease of visual recall, by the user, those beadsets selected for use in an experiment. Optional virtual arrow buttons585 located on the Bead Set tab 570 between the Available option areaand the Selected option area provides means for adding or removing, by auser, singularly or as a group, the bead sets to be used. For example,the arrow buttons 585 optionally includes a single right arrow buttonand a single left arrow button for add on selected bead set and removeone selected bead set, respectively. In addition to or alternatively,for example, a double right arrow button and a double left arrow buttonare optionally implemented to add all bead sets to the “Selected” optionarea and to remove all bead sets from the Selected Option area,respectively.

The Bead Set tab 570 optionally includes a Caption option 590 havingmeans for labeling or renaming beads, by a user, to correspond to aspecific assay or function, for example, for ease of user recognition.Optionally, in the selected option area or window, an original numericor alphanumeric designation of the bead is optionally included with alabel or name provided by the user, for example, using the Captionoption 590.

The Bead Set Tab 570 includes a Minimum Distribution (%) option 595.This option includes means, such as a virtual slider, for selecting, bya user, a minimum distribution level between, for example, 1 and 99%that allows the instant system to disregard, for example, any bead setthat does not collect enough events to be statistically significant. Forexample, if the instant system is set up to collect 1600 events persample and 16 bead sets are included in each sample, then a mean of 100events per bead set is expected. The user optionally decides the minimumnumber of events per set which is statistically acceptable. That is, forinstance, if all but one bead set register between 85 and 120 events andone bead set registers 7 events, it is likely that a problem exists withthat one bead set. However, by setting the Minimum Distribution (%)option 595 to, for example, 25%, each set must register 25 events (25%of 100 beads per set) or it will be discarded from the data collection.As such, data optionally is not collected and/or displayed for a beadset that falls below the desired minimum distribution level.

The Bead Set tab 570 optionally includes a “Normalize FluorescenceChannel 1 (FL1)” option 600. This option includes a user-selection entryarea or checkbox and means for prompting the system to eliminatespurious data points that could throw off the data as a set, when thischeckbox is selected or clicked, for example.

The Bead Set tab 50 optionally includes a “Background Control Sample”option 610. I have discovered that the dyes contained within standardmicrospheres or beads bleed to some degree into the Fluorescence Channel1, the channel arbitrarily chosen to reflect the detected fluorescenceof the analytes of interest. This spectral overlap, or bleeding,optionally, is advantageously corrected in the Histogram frame on theCompensation tab by using, for example, a FL1-% FL2 setting ofapproximately 40%.

However, I determined that a more accurate method of this spectraloverlap correction includes recording the mean sample spillover of eachbead type in the absence of any analyte and reporter. This meanspillover is optionally subtracted from the later sample or samples meanto obtain the samples' true mean fluorescent readings.

The “Background Control Sample” option 610 includes a user-selectionentry area or checkbox, for example. Checking or selecting the“Background Control Sample” checkbox 610 activates this option.Optionally, as a default measure, if this checkbox is not selected orchecked, compensation is optionally still adjustable via theCompensation tab 485. When this option is so activated, the first samplein the Results Table 370, for example, reads “Background” and, forexample, is optionally not able to be renamed once set. After runningthe sample, the user optionally saves the results as a template forfuture experiments, thereby allowing reuse of these spectral spillovervalues in future folders without running a background sample first. Toensure the greatest possible accuracy, optionally, the background sampleadvantageously is run with each experiment, and advantageously aftercalibration or changes to the optical amplifier or PMT's settings.

The Multiplexed Analysis application module 305 optionally includes, forexample, a Permanent Bead Grid in Dot Plot Display 535. As mentionedabove, each bead set, used in accordance with the instant invention,advantageously has a unique spectral region in the Fluorescence Channel2, Fluorescence Channel 3 (FL2×FL3) dot plot that advantageouslycorresponds to the bead sets's user-selected or manufacturer-selectedunique numeric or alphanumeric designation. The region, for example, ispre-defined. If the bead sets selected for use do not fall in theirrespective regions, the user is advantageously alerted to the need torecalibrate the flow analyzer. The Dot Plot Display 535 includes meansof showing the results of a sample run, displaying all gated events, forexample, all events passing through the side scatter gate.

The Dot Plot Display 535 optionally includes a Show Bead option 615.This option, for example, includes a pull-down menu and means forviewing all bead sets (gated and ungated), all gated events only, and/ora specific bead set by selecting, by the user, from an all eventsoption, an all gated events option, and/or a bead number option from thepull-down menu. By way of example, the all events option includes meansfor showing all beads, gated and ungated, and includes events outsidedesignated bead set region. The all gated events option includes meansfor showing events registering in, for example, the side scatter gateand includes events outside the designated bead set regions. The beadnumber option includes means for showing up to all data associated withthe bead type selected. Thus, data is optionally discriminated by, forexample, the side scatter gate and the bead type region.

Optionally, the Dot Plot Display 535 includes means for viewing specificbead data by clicking or selecting, by a user, in the desired bead typeregion on the Dot Plot Display 535. The Dot Plot Display 535 optionallyfurther include means for displaying Bead Details for that bead type in,for example, a box or tabular format. For instance, the box optionallydisplays the original bead type alphanumeric code, the user-selectedbead type name, and/or the count, i.e., the number of events collectedin that bead type region.

Assay Development Overview

Assays, in accordance with the instant invention, are set up in anystandard configuration for standard binding assays. For example, directbinding of a fluorescent molecule, capture/sandwich assays with afluorescent “secondary” antibody, competitive inhibition assays with afluorescent ligand, and/or DNA hybridization assays and enzymatic assaysare performed. By way of illustration, set up of an acceptable assayinclude the following. A target molecule is coupled- to each bead in abead set. A reporter molecule is labeled with, for example, a greenfluorescent reporter group. The assay is optionally optimized, forexample, for concentrations of target and reporter molecules, numbers ofbeads, and/or assay conditions.

As to target coupling, sample procedures for coupling, for example,proteins or oligonucleotides to beads of a given subset are as follows.These procedures are intended only as non-limiting, exemplary guidelinesfor initial assay setup. By way of illustration, the guidelines includecoupling of near-maximal amounts of target molecules to the beads.Alternatively, the guidelines include coupling of minimum detectablequantities of target molecules to the beads.

Sample Method of Operation

In view of the above-described apparatus, a method of operationtherefor, given by way of illustration only, is provided herewith. Asindicated above the beads are identified into their respective, separatesubsets. Each has a bead identifier (ID) associated with it. For eachbead ID, for example, fluorescence channel 1, FL1 statistics arecollected. It is the FL1 statistics that the researcher or the clinic isgenerally interested in, because it is an indicator of how muchbiological activity is seen on the bead. The beads include a set amountof Fluorescence channel 2, (FL2) and Fluorescence channel 3 (FL3). But,it is in an individual sample, e.g., the human serum, whereinreactant-analyte interaction defines how much of the FL1 signal orfluorescence is found or detected.

For example, a hundred events of each bead type are collected. However,the assay is what is to be read. For those one hundred events, all ofthe FL1 signals are summed, and an average or peak reading or standarddeviation, coefficient of variation, and a variety of statistics aretaken therefrom. Once the statistics have been collected, the softwareapplication generates a data table, the rows representing the differentsamples that have been run.

While the software and hardware are running, the events come in and areoptionally displayed in real time on, for example, OCX graphic controls,such as described above and illustrated in the figures. In general, anOCX is an Object Linking and Embedding (OLE) custom control, aspecial-purpose program that can be created for use by applicationsrunning on Microsoft's Windows systems. Advantageously, use of OLEsupports the development of “plug-and-play” programs that can be writtenin any language and used dynamically by any application in the system.These programs are known as components, and the application in whichthey are run is known as a container. In the instant invention, the OCX,for example, are responsible for displaying, for example, histograms forany one of the channels. Thus, the control and analysis software 50 has,for example, two controls which are OLE controls. The control andanalysis software 50 instructs a graphics component called “LumGraphcomponent,” or LumGraph.OCX 700, to behave, for example, as a histogramor to behave, for example, as an X-Y plot.

A LumAPI application program interface component 705, when it gets themachine information in real time from the flow analyzer, for example,spews things out to a file, as shown schematically in FIGS. 43 and 44.An API, or application program interface, is the specific methodprescribed by a computer operating system or by another applicationprogram by which a programmer writing an application program can makerequests of the operating system or another application. Advantageously,the LumAPI component 705 includes a dynamic link library (DLL). A DLL isa collection of small programs, any of which can be called when neededby a larger program that is running in the computer.

The small program lets the larger program communicate with a specificdevice such as a daughter board, which is often packaged with a DLLprogram (usually referred to as a DLL file). Advantageously, the LumAPIcomponent 705, optionally implemented as a DLL file, need not be inrandom access memory (RAM) together with the main program, therebysaving RAM space in the computer. As shown in FIG. 43, the control andanalysis software 50 links the LumGraph component 700 and the LumAPIcomponent together 705, for example, by telling the LumGraph component700 what the real-time output is going to be. The LumGraph component700, for example, then accesses this real time output. Then, theLumGraph component 700 outputs the data into these displays which areavailable via control and analysis software 50. The control and analysissoftware 50 initializes the LumAPI component 705, and tells it whatmachine to talk to and for which bead types to look. It has, forexample, sixty-four possible bead types. Other numbers of bead typesare, of course, acceptable.

The LumAPI component 705, for example, stores in a database 720 anoutput of events, and raw data representing upper channels, sidescatter, FL1, FL2, and/or FL3. The LumGraph component 700, for example,accesses the same data in a graphical way for presenting into thecontrol and analysis software 50, which then displays the data.

Statistics are, for example, generated in the LumAPI component. Thecontrol and analysis software 50 retrieves the statistics from theLumAPI component 705, and re-polls it, periodically or aperiodically, toobtain the latest statistics. Thus, the statistics go back to thecontrol and analysis software 50, which displays them appropriately, forexample, in a table in real time. The software 50 performs statisticalanalysis, for example. It then optionally records any results of thestatistics to one, two, or more external file types 710 and 712, forexample, in a spreadsheet format.

As to the apparatus components, it is to be understood that the testprobe, or needle, for example, enters the sample tube to draw the sampleout of the tube and into the machine. For example, the syringe pumpactually goes down and draws that sample in through a valve. The valveoptionally includes a three way valve in the syringe pump, so that afterthe sample is drawn into a standard sample loop. Then, the valve isswitched over to inject the sample through another tube into the opticalassembly. Advantageously, the inclusion of the sample loop preventssample fluid from contaminating the syringe pump. The sample, forexample, goes into the optical assembly through a needle, the dropletsforming at the tip of that needle.

As mentioned above, the droplets comprise the beads and solution. Thereare, for example, a billion beads, or more or less, in a tube. The beadsare, for example, so small that one cannot see them with the naked eye.Indeed, the mixture of the beads and the solution, for example, lookslike clear water. The droplets are formed, for example, at the tip ofthat needle inside the cuvette on the optical assembly.

As mentioned above, the system includes a sheath fluid container holdingthe sheath fluid supply, such as a water supply. A compressor pumpprovides air pressure over the top of the water, forcing the water intothe system at, for example, approximately 5.5 psi. A pinch valve shutsoff that water supply, when samples are not running so as to conservewater. The air pressure drives this water into, for example, the verysame optical assembly after it passes the pinch valve. There, it fillsthe cuvette up to the neck down region of the cuvette, which resemblesan inverted half of an hour glass.

Sheath fluid, e.g., water, fills up the large portion of the cuvette andas the water gets forced into the neck down region, the wateraccelerates very rapidly. So, instead of moving at about, for example, atenth of meter per second, the sheath fluid moves at, for example, aboutfive meters per second in that neck down region just because of thevolume restriction. The water accelerating causes the sample drop thatforms on the end of the needle to be elongated. The hydro-dynamicprinciples behind this are well known. By the time the fluid arrives atthe area of the optical assembly where the lasers are, the sample hasbeen stretched into a very tiny strand that is, for example, abouttwelve microns in diameter. Of course, other sized strands, larger orsmaller are also effectively used.

At the viewing area, there is a two hundred micron chamber or capillary,having, for example, a 200 micron cross-section. The outer 188 micronsof the cross-section contains water, and the very center orsubstantially center of the cross-section, for example, the inner most12 microns, contains the sample. The optical sources, such as lasers,are aligned, or pointing, to a precise point along the capillary fromfor example, substantially opposites sides, concentrating a beam, forexample, about 30×60 microns wide and long under the same spot. As thebeads and the sample fluid pass through the laser beam, the beads, whichhave fluorescent dye inside, are illuminated and then they startfluorescing, which means that they emit a longer wavelength at whichthey were excited.

Optical detectors surround the viewing area or chamber, and also pointto that very same spot where the lasers pointed. The optical detectorssubstantially focus on the same spot and image the bead as it is passingthrough the light beam. That is, the fluorescence values correspondingto, for example, fluorescence channels, 1, 2, and 3, detected aretransmitted to a fiber optic cable connected to each or all of theoptical detectors. The cable optionally includes a multi-mode fiber,wherein the light travels down the fiber and through an optical filter.The optical filter, for example, only allows certain wavelengths ofinterest to pass through into the electronics, for instance, fourfilters, four channels, and four detectors.

After the light in the band of interest passes through the filters, itenters an optical amplifier, such as a standard avalanche photodiode. Anavalanche photodiode is a circuit that converts light or photons intoelectrons. The more light that goes into an avalanche photodiode, themore current is admitted by the avalanche diode. That current is thenconverted to a voltage by a transmit beads amplifier with a gain of, forexample, about a million times, i.e., re-amplified a million times, forexample. The current is then inputted to an op-amp filter that bandlimits the signal to eliminate substantially all high frequency noiseto, for example, about 450 kHz. The output of the op-amp optionally isthen amplified one more time before it goes into, for example, one offour A to D converters. Alternatively, a greater number of A to Dconverters are also possible and is limited only by the processing powerof the associated central processing unit. For example, five or eight Ato D converters are optionally included. On the back side of each A to Dconverter, is, for which reads each of the four A to D converters,thereby performing, for example, four million read operations everysecond, i.e., one million read operations for each of the A to Dconverters. Plainly, if, for example, five A to D converters wouldentail five million read operations every second. It stores it in one ormore circular buffers inside the DSP's memory.

Another thread of the DSP's operation constantly searches the sampledata for the presence of an event. An event includes signal levels abovethe background level, and appears as one or more pulses created by theavalanche photodiode in increasing numbers via the A to D converters tothe DSP. When the number gets above the certain threshold, optionallyset by the user, an event is triggered and that pulse is analyzed by theDSP software. Optionally, and advantageously, if it is a single beadevent as opposed to two beads passing through the beam at the same time,the DSP accepts that as a valid event, based again by analysis of itswave form. The values for all the fluorescence channels, the peak valuesfor each fluorescence channel, are then passed on in record form to, forexample, a micro controller.

The micro controller, which is advantageously linked to the DSP throughdirect memory access, takes these packets out of the DSP memory, formatsthem, and passes them onto the control and analysis software. The microcontroller is also responsible for a number of other functions. Itoptionally controls the syringe pump, for example, telling it how muchto draw, how fast to draw, and/or whether to expel waste. It optionallycontrols performance of a wash cycle on the syringe pump to preventsample carryover, for instance. The micro controller optionally controlsthe pinch valve which starts and stops the sheath flow into theinstrument. It optionally controls the high voltage bias of theavalanche photodiodes. The avalanche photodiodes optionally require veryhigh voltage to operate, and so the micro controller optionally sets theprecise voltage for each fluorescence channel based on the properties ofeach particular avalanche photodiode. An avalanche photodiode optionallyneeds as little as 10 volts in order to operate at minimum efficiency oras much as 200 volts to operate at maximum efficiency. The microcontroller optionally regulates this operating voltage also. Itoptionally also senses a switch when the aspirator arm is down to knowto begin drawing sample. The micro controller optionally also monitorsthe air pressure to ensure sufficient air pressure on the sheath fluidto provide a valid sample of, for example, 12 micron core size.Obviously, the lower the pressure on the sheath fluid, the larger thecore size, whereas the higher the pressure, the smaller the core size.

Advantageously, at sheath fluid pressure of 6 psi to 7.5 psi, forexample, approximately 6.5 psi, and an injection rate of the sample atapproximately 1 microliter per second, the desirable 12 micron samplecore is obtained. It also monitors the high voltage and reads it back tomake sure that everything is operating properly and does numerousdiagnostics. The top of the system optionally includes an air cylinderthat optionally shares the same air pressure pump that drives the sheathfluid through the system. The extra pressure is used to move the aircylinder up and down. The air cylinder is optionally attached to thetest probe. Consequently, by forcing air either into the top or thebottom of the air cylinder, the sample needle is moved up and down. Sucha configuration advantageously reduces the operator's responsibilitylargely to pressing the start button to “on” in the instant diagnosticsystem.

There are several advantages of this overall system. The light sourcesetup, such as the laser setup, is very stable since all components aremounted to the same base, permitting a smaller or tighter spot beam forthe lasers. That is, because the setup is so stable, a fairly accuratespot on the fluid flow stream is obtained for reading the beads. Anotherfeature of the present invention is that as a result of the stability,low power lasers are used, such as those having 10 milliwatt powerrequirements. However, lasers having power requirements less than orequal to 3 milliwatts up to 30 milliwatts are alternatively used.

Another feature of the present invention is that a compressor withfairly low psi rating is used relative to other cytometers that requirevery expensive compressors with fairly high psi ratings. Fast cytometersthat can run upwards of 20,000 beads per second usually require a lot ofpressure, such as 30 psi and up. So, the compressor being used in theinstant invention is much less expensive, more compact, and longerlasting than prior flow cytometers.

Another important feature of the invention is that the optical assemblyis made of many different pieces, but are optionally bolted togetheronto one solid piece that holds the cuvette, the viewing chamber, thelasers and the detectors, all securely mounted together. Advantageously,the assembly comprises stainless steel, although other sturdy materialsmay also be used. In addition, the laser/detector assembly is compact,durable, and is easily shipped with little or no functional damage.

Sample Program Component

A LumAPI Library 705 includes, for example, a standard applicationprogramming interface library, which communicates with the flowcytometer via the serial or parallel connection. The LumAPI Library 705optionally includes standard communication functions, such as shown, byway of example, in FIG. 44.

A standard MHMath Library 715, such as shown in FIG. 43, includesroutines for calculating a polynomial trend-line, using an arbitrarynumber of data points, for example, one, two, three, or more, for input,and for calculating a polynomial of any order. The algorithms thereinare, for example, derived from C/C++ Mathematical Algorithms forScientists and Engineers, Namir C. Shammas, ©1995, McGraw-Hill,incorporated herein by reference.

As shown in FIG. 43, the control and analysis software 50 communicateswith either or both of these libraries. It includes a main userinterface program, which includes the GUI between a user and the instantinvention. This program or diagnostic system application is developedusing, for example, Microsoft Visual Basic or any other suitableprogramming language. It optionally includes visual components, such asscreen displays, user input facilities, such as dialog boxes, and/oruser option facilities, such as, for printing. The diagnostic systemapplication also includes a controlling program to communicate with theLumAPI library 705, which in turn communicates to the flow analyzer viathe serial or parallel connection.

The diagnostic system application includes an initialization means orcomponent. The initialization component includes, for example, a LumInitcomponent 725, which initializes the device interface for the flowcytometer to use flow cytometer resources. The LumInit component 725,for example, includes one or more standard callable software functions,discussed below, which are called prior to calling another LumAPIfunction. The initialization component, for example, returns a non-zeroreturn value indicating an occurrence of an error during initialization.In such a case, a default return value, for example, is zero. The stepsperformed by the initialization component includes one or more of thefollowing steps.

Initialization Functions

LumInit Component

A LumInit component means 725, or function, for example as shown in FIG.44, as mentioned above, initializes the device interface for the flowcytometer. Optionally, the LumInit component 725 is one of the availableLumAPI functions. Advantageously, the control and analysis software, forexample, calls it prior to calling any other LumAPI function. TheLumInit component 725 includes initializing a multimedia timer forbackground processing to poll the DSP, for example, by:

starting the background task;

calling a flow cytometer initialize function, which includesinitializing relevant data structures;

calling an instrument reset function to initialize the data structures,to load the DSP binary values into memory, to reset the DSP andoptionally, pause to load the DSP binary to the serial or parallelconnection interface board and look for an initialization “O.K.” status,to set an error status if the initialization status is not “O.K.”, andto notify the DSP of the operational status of the computercommunicating therewith; and/or

initializing flow cytometry standard values.

Once finished with using the LumAPI library 705, the control andanalysis software 50 calls, for example, a LumTerminate component orfunction 730.

LumTerminate Component

A LumTerminate component means 730, or function, for example as shown inFIG. 44, closes a device session with a flow cytometer, thereby freeingany flow cytometer resources used created by calling the LumInitfunction 725. Optionally, the LumTerminate component 730 is a LumAPIfunction. The LumTerminate standard functions, for example, include:

killing the background task;

freeing memory; and/or

calling an instrument terminate function.

LumSet Bead Map File Component

A LumSet Bead Map File component means 735, or function, such as shownin FIG. 44, loads a file, which defines, for example, a two-dimensionalbead map used to distinguish one bead type from another, i.e., beads ofone bead subset from those of another. Optionally, the LumSet Bead MapFile component 735 is a LumAPI function. By way of example, if only onefluorescent dye is used per bead type, a one-dimension bead map is used.

LumReset User Beads Component

A LumReset User Beads component means 740, or function, for example asshown in FIG. 44, is the first function called when a user is definingwhich beads an assay will be using. Optionally, the LumReset User Beadscomponent 740 is a LumAPI function. The LumReset User Beads function740, for example, resets an internal table of user beads in the LumAPIlibrary 705.

Optionally, a user makes repeated calls to a LumSet User Beads function745 to build a list of bead types needed for an assay. Alternatively, inan alternative embodiment of the function, the user makes a single callto the LumReset User Beads function 740 to build the list of needed beadtypes. Optionally, the LumReset User Beads function 740 is omitted if,for example, the LumSet User Bead function 745 discussed hereinbelow,can write over values stored in the LumAPI's table of needed bead types.

LumSet User Bead Component

A LumSet User Bead component means 745, or function, such as shown inFIG. 44, informs the LumAPI library 705 that a user will be interestedin acquiring bead statistics for a bead by a unique identifierassociated with the given bead type. Optionally, the LumSet User BeadComponent 745 is a LumAPI function.

The user, for example, makes repeated calls to the LumSet User Beadfunction 745 to add additional beads to the list of interested beadtypes. In an alternative embodiment, the user makes a single call to theLumSet User Bead function 745, add additional beads to the list ofinterested bead types. The user, as discussed above, calls the LumResetUser Beads function 740 prior to calling the LumSet User Beads function745 to clear the internal list of beads. Optionally, a parameter in theLumSet User Bead function 745 includes a user-supplied literal to beassociated with the given bead type.

DSP Control and Monitoring Components

LumRead Panel Settings Component

A LumRead Panel Settings component means 750, or function, such as shownin FIG. 44, copies a current set of flow analyzer settings into auser-supplied buffer. Optionally, the LumRead Panel Settings component750 indicates which, if any, of the settings have changed since aprevious call to the LumRead Panel Settings function 750.

LumChange Panel Settings Component

A LumChange Panel Settings component means 755, or function, such asshown in FIG. 44, allows a user to change one or more flow analyzersettings at a time. The settings in a user-supplied buffer, which holdsthe desired panel settings, are optionally modified beginning with thefirst parameter in the supplied settings which needs changing and endingwith the last parameter in the supplied settings which needs changing.

Sample Acquisition and Result Reporting Components

LumStart Test Component

A LumStart Test component means 760, or function, such as shown in FIG.44, tells the LumAPI library 705 to begin acquiring bead statistics forthe current sample loaded on the flow analyzer using, for example, abackground task. Prior to calling the LumStart Test function 760, thesample fluid to be analyzed is loaded into the flow analyzer, and theflow analyzer is placed in the RUN mode. Alternatively, the sample fluidloading and flow analyzer running is automatically operated by theinstant software, either by the LumStart Test component 760 or anoperatively linked function.

LumStop Test Component

A LumStop Test component means 765, or function, such as shown in FIG.44, ends the acquiring of bead statistics for the current sample.

LumQuery Test Results Component

A LumQuery Test Results component means 770, or function, such as shownin FIG. 44, copies the most current bead statistics into a user-suppliedbuffer. Bead statistics are contained, for example, in a user-suppliedtable of test result data structures. The table is large enough tostore, for example, less than 100 test result data structures, 100 suchdata structures, or more than 100 such data structures.

The LumQuery Test Results component 770 is called at any time after theLumStart Test component 760 is called to get substantially immediatestatistics prior to collecting all of the requested beads. In analternate embodiment, the LumQuery Test Results component 770 is calledprior to or concurrently with the LumStart Test component 760, providedthat the LumQuery Test Results component 770 includes a triggercomponent. That is, the LumQuery Test Results component 770 isoptionally dormant, until operation of the LumStart Test component 760triggers activity of the LumQuery Test Results component 770.

LumQuery Test Total Component

A LumQuery Test Total component means 775, or function, such as shown inFIG. 44, returns data acquisition statistics.

I have recognized that near-maximal levels of target molecules are notoptimal for all assays, depending on the desired properties. Forexample, capture/sandwich assays are more sensitive with high levels ofcapture molecules per bead than with high levels of target molecules perbead. As another example, I have determined that inhibition assays aremore sensitive with limiting amounts of capture molecules per bead thanhigh levels of capture molecules per bead.

As to reporter labeling, I have recognized that amine-reactivederivatives, for example, of green fluorescent dyes, for example, arewell-suited for labeling reporter molecules. Optionally, the same dye isused for all assays multiplexed together. Alternatively, different dyesare used for one or more of the assays multiplexed together. By way ofexample, fluorescent reporter molecules are prepared by substitutionwith, for instance, green fluorescent dyes. The BODIPY® dye by MolecularProbes, Inc., for example, is an acceptable green dye for use in assays,according to the instant invention, and, in particular, for preparationof reporter molecules. Alternatively, fluoroscein-labeled reportergroups are also used with a higher spectral overlap compensation settingfor “bleed” into, for example, the orange channel than required forBODIPY®-labeled reporter groups. For example, whereas an FL2-% FL1compensation for BODIPY® is, for instance, set to approximately 20, anFL2-% FL1 compensation for fluoroscein is, for instance, set toapproximately 34. FL2-% FL1 compensation for any other green dye, forexample, is determined empirically by measuring the percent of anyspectral overlap of the green fluorescence (FL1) channel, for example,into the orange fluorescence (FL2) channel, for example, using standardassay development beads.

By way of illustration, virtually any protein or peptide can be labeledusing the BODIPY-FL-CASE dye by Molecular Probes, Inc. Proteins orpeptides can also be labeled with fluoroscein derivatives, such asfluoroscein isothiocynate (FITC). Synthetic oligonucleotides of, forexample, 15 to 40 bases can be successfully used for hybrization assaysconsistent with the instant invention. Oligonucleotides having more thantwo bases but less than 15 bases, or greater than 40 bases are alsoacceptable. For example, complementary A and B strands are required foreach genetic sequence to be analyzed. That is, one strand is coupled tothe target beads, and the other strand is conjugated to, for example, agreen fluorescent reporter dye. For example, each oligonucleotideincludes a standard spacer and/or linker between the terminal nucleotideand the amino group. To this extent, for example, a C9-spacer and, forexample, a C6-amino-terminal linker are used during synthesis. Thesynthesis, for instance, results in a total length of, for example,approximately fifteen atoms. Fewer or more atoms in the spacer are alsopermitted. However, for instance, a total spacer instance of less thantwelve atoms reduces the performance of the hybridization assays.Oligonucleotides are connected during synthesis, for example, either atthe 3′ or the 5′ end. I have recognized that 5′ connections are usuallyless expensive than 3′ connections.

In general, the concentration of target molecules per bead used in thebead coupling reaction are optimized, for example, by filtration in thesame way that a coating of a microfilter plate is optimized. Optionalconcentrations of reporter molecules per bead are determined, forexample, by filtration with a fixed number of beads. Optionally, theextent of fluorescent labeling of these molecules are also varied.

I have determined that the number of beads used in an assay affect theamount of target molecules present and/or the analysis time on the flowanalyzer. Specifically, I recognized that greater numbers of beadsresult in more sensitive assays. Using, for example, 1,000 to 10,000beads per assay allows for analysis in, for example, less than onesecond, one second, two seconds, three seconds, or up to a minute.However, the number of beads is optionally increased or decreased toimprove sensitivity or decrease run time, as desired.

Assay conditions for assays according to the instant invention, forexample, are similar to conditions for reactions performed in otherstandard formats. That is, for example, immunoassays are performed in,for example, a buffer which limits non-specific binding, such as PBScontaining approximately 0.1% BSA. As another example, hybridization ofnucleic acids are performed in most standard hybridization buffers. Whencomplex mixtures of sequences with disparate guanine and cytosine pairsare present in multiplexed assays, a buffer system, such as tetramethylammonium chloride (TeMAC), which minimizes differences in melting pointsare used advantageously. Because guanine and cytosine pairs includethree hydrogen bonds between the bases, and adenine and thymine pairsinclude two hydrogen pairs, a DNA strand having a higher concentrationof guanine and cytosine pairs than adenine and thiamine pairs will havea higher melting point than vice versa.

The many features and advantages of the invention are apparent from thedetailed specification, and thus, it is intended by the appended claimsto cover all such features and advantages of the invention which fallwithin the true spirit and scope of the invention. Further, sincenumerous modifications and variations will readily occur to thoseskilled in the art, it is not desired to limit the invention to theexact construction and operation illustrated and described, andaccordingly, all suitable modifications and equivalents may be resortedto, falling within the scope of the invention.

GLOSSARY API Application Program Interface, a set of functions usuallyin a separate library which the calling program can use. Bead seeMicrosphere Channel Channel can have two similar but separate meanings.A channel normally is synonymous to any PMT reading associated with agiven light scattering event. Thus as an object passes through thelaser, multiple channels report their readings including ForwardScatter, Side Scatter, FL1, FL2 and FL3. The alternate meaning refers tothe form of data associated with a channel. For the FACScan, channeldata is reported as a 10 bit reading, thereby producing a number from 0to 1023, or 1024 different “channels”. The application has the option ofreporting the data in its raw form of “channel data” or in a logarithmicmode from 0 to 10⁴ −1. For example, if the documentation describessomething as having “only 10 channels of difference,” then it isreferring to the data for a given channel as it comes into the computerand not to different PMTs. Doublet A clumping of two or moremicrospheres. Doublets produce more scattered light signal than singlebeads and thus can lead to incorrect analysis. Doublets are rejectedfrom analysis by employing a side scatter gate. DSP Digital signalprocessor. It is a standard computer processor chip capable of very fastmathematical operations. FL1 Fluorescence channel one. It is designed tocapture only light of a given color, e.g., green. The light first passesthrough a wavelength filter and is then collected on the FL1 PMT. FL2Fluorescence channel two. It is designed to capture only light of agiven color, e.g., orange. The light first passes through a wavelengthfilter and is then collected on the FL2 PMT. FL3 Fluorescence channelthree. It is designed to capture only light of a given color, e.g., red.The light first passes through a wavelength filter and is then collectedon the FL3 PMT. Forward Scatter This refers to the amount of lightpassing directly through the patient's sample at a given instance.Forward scatter normally provides a measurement for the size of anobject passing through the laser beam. GAM Refers to a test of IgG, IgAand IgM human antibodies. These antibodies are usually present to attachthemselves to an infectious agent present in the body so that it islater destroyed. Gating Gating refers to a method of filtering outevents by the application. For purposes of the instant invention, gatinginvolves only allowing events, which belong to a narrow, predefinedrange in the side scatter channel. This ensures only objects of the sizeof a single microsphere are collected. GUI Graphical User Interface, aprogram that is used to interface to the operator. IgG The IgG antibodyshows up after the IgM antibody and will adapt to provide lifelongimmunity to an infectious agent. IgA The IgA antibody is normally foundin the salivary glands. IgM The human IgM antibody is normally the firstantibody to attack an infection and will be present for the first twoweeks of an infection. Microsphere Precisely manufactured 5.5 microndiameter spheres with a tolerance of ±0.1 micron. Spheres of other sizesare acceptable. MIF Mean Intensity of Fluorescence (a.k.a. MFI—MeanFluorescence Intensity) PMT Photo multiplier tube. Located inside theflow cytometer, it amplifies low levels of light and provides a methodfor digital conversion. A given wavelength of light can be measured bysupplying a light filter. Sheath Fluid A relatively rapid streamconsisting of what is usually highly filtered water. By having thesheath fluid flow much faster than the injected patient sample, thepatient sample drawn toward the center of the combined streams. Thisenables the patient sample to pass through the center of the focusedlaser beam. Side Scatter This refers to the amount of light that hasbeen deflected at a right angle to the direction of the laser beam. Sidescatter provides an alternate measurement of the relative size and shapeof the object passing through the laser beam. Singlets A singlemicrosphere, one which is not attached to another microsphere. Seedoublets.

1. A multi-analyte diagnostic system for use with a computer, thediagnostic system comprising: a flow analyzer including a substantiallyco-planar optical assembly having at least one light source and at leastone optical detector, said flow analyzer being communicable with thecomputer; and a memory medium readable by the computer and storingcomputer instructions, the instructions including: processing abiological sample using said flow analyzer, wherein said processinginstruction includes: (a) exposing a pooled population of subsets ofparticles to the biological sample, the particles in each subset having(i) at least one classification parameter that distinguishes theparticles of one subset from those of another subset, and (ii) areactant specific for each of at least one analyte of interest; and (b)passing the exposed pooled population of subsets of particles through anexamination zone; and determining a presence and quantity of each ofsaid at least one analyte of interest in the biological samplesubstantially simultaneously to said processing step, wherein saiddetermining instruction comprises assessing the identity and quantity ofeach of said at least one analyte of interest, if present, in the sampleby substantially contemporaneously: (a) collecting data relating to theat least one classification parameter, including particle subset data onfluorescence emission intensities; (b) collecting data relating to apresence or absence of a complex formed between the reactant and ananalyte of interest specific to the reactant, including analyte data onfluorescence emission intensities, wherein the particle subset data andthe analyte data exhibit spectral overlap; (c) classifying, withoutrelying exclusively, if at all, on differences in particle size, eachparticle according to its subset, wherein said classifying step includesreducing the spectral overlap sufficiently to identify said eachparticle according to its subset; and (d) quantifying an amount ofcomplex associated with each subset.
 2. The multi-analyte diagnosticsystem according to claim 1, wherein said flow analyzer furthercomprises: a plurality of light sources and a plurality of opticaldetectors, said plurality of light sources including overlapping focalregions; and a plurality of magnification lenses for magnification oflight emissions or reflections detected by said at least one of theplurality of optical detectors each of the plurality of magnificationlenses having a magnification factor of at least 15X.
 3. Themulti-analyte diagnostic system according to claim 2, wherein saidplurality of light sources includes a plurality of laser diodes emittingcontinuous wave light.
 4. The multi-analyte diagnostic system accordingto claim 3, wherein said plurality of laser diodes includes laser diodesemitting a plurality of wavelengths of continuous wave light.
 5. Themulti-analyte diagnostic system according to claim 1, wherein said flowanalyzer includes a cuvette having a flat air-to-glass interface.
 6. Themulti-analyte diagnostic system according to claim 1, further comprisingat least one of a vertically and horizontally moveable platform, whereinsaid flow analyzer further comprises a vertically moveable aspirator,said platform cooperating with said aspirator.
 7. The multi-analytediagnostic system according to claim 6, wherein said platform supportsone of a microtiter plate and said flow analyzer.
 8. The multi-analytediagnostic system according to claim 1, wherein said flow analyzerfurther comprises an aspirator moveable in either one of vertical andhorizontal directions.
 9. The multi-analyte diagnostic system accordingto claim 1, further comprising a circular memory buffer communicablewith said flow analyzer.
 10. The multi-analyte diagnostic systemaccording to claim 9, wherein said circular memory buffer includes afirst movable pointer in operation, pointing to a storage positionavailable for storing new data, and a second movable pointer, inoperation, pointing to a storage position having unanalyzed data. 11.The multi-analyte diagnostic system according to claim 1, wherein thecomputer instructions further provides at least one of: a main menu; aresults table; a system monitor; a dot plot display including at leastone of a density dot plot and a decaying dot plot; a histogram tab; anoptical amplifier control tab; a color compensation control tab; and adoublet discriminator control tab.
 12. The multi-analyte diagnosticsystem according to claim 5, wherein said cuvette includes asubstantially flat glass-to-fluid interface.
 13. The multi-analytediagnostic system according to claim 12, wherein said cuvette includes aneck region having one of an internal rectangular cross-section and aninternal square cross-section.
 14. The multi-analyte diagnostic systemof claim 2, wherein at least one of the plurality of magnificationlenses has a magnification factor substantially within a range between20X and 40X.
 15. The multi-analyte diagnostic system of claim 14,wherein two of the plurality of magnification lenses each has amagnification factor substantially equal to 25X.